Oncolytic Herpes Simplex Virus And Therapeutic Uses Thereof

ABSTRACT

The present invention relates to variants of herpes simplex virus (HSV) that selectively infect and replicate in cancer cells, including HSV strains that selectively infect and replicate in bladder cancer cells. Preferred HSV of the invention have intact endogenous Us11 and Us12 genes and have genes encoding ICP34.5 replaced with a gene encoding Us11 fused to an HSV immediate early (IE) promoter. The variant HSV of the invention also comprise one or more additional heterologous genes encoding immunomodulatory polypeptides. Methods and compositions using these variant HSV, e.g., for treating cancer in a subject, are also provided.

RELATED APPLICATION

This application claims priority to U.S. Provisional application Ser.No. 61/532,335, filed Sep. 8, 2011, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to avirulent, modified herpes simplexvirus (HSV) that replicates selectively in cancer cells, such as bladdercancer and melanoma cells. Therapeutic methods using the modified HSVare also provided, including therapeutic methods for treating bladder,melanoma and other types of cancer.

BACKGROUND OF THE INVENTION

While the underlying goal of cancer therapy is to destroy the cancerwhile avoiding excessive damage to the normal organs of the body, theirtoxic effects to the body limit present treatments such as chemotherapyand radiation. As such, the maximal tolerable dosage of such therapiesis often inadequate to eradicate the tumor. Newer treatment strategieshave focused upon identifying antineoplastic agents that can distinguishnormal cells from their cancerous counterparts. Oncolytic virusesreplicate, spread and selectively destroy cancerous tissue, but areattenuated and do not harm normal cells. In addition to directoncolysis, an immune-mediated component contributes to oncolytic virusefficacy in immune-competent mice (i.e., oncolytic viruses have atumor-vaccination effect mediated at least in part through an anti-tumorCD8+ T cell response). Using immune-competent mice with syngeneic,bilateral subcutaneous (s.c.) tumors, previous studies established thattreatment of one tumor with oncolytic virus (HSV-1) induced regressionof the treated and untreated contralateral tumor (see Toda M, et al.“Herpes simplex virus as an in situ cancer vaccine for the induction ofspecific anti-tumor immunity.” Hum Gene Ther 1999; 10:385-93). Whiletreated and untreated tumors both regressed, oncolytic virus was onlydetected in the treated tumor. Furthermore, regression of theuninjected, contralateral tumor resulted from an anti-tumor CD8+ T-cellresponse.

Several different oncolytic herpes simplex virus type 1 (HSV-1) strainshave proven to be safe in phase I human clinical trials. See Aghi &Martuza, Oncogene (2005) 24:7802-7816. Viral genetic analysis hasestablished that HSV-1 can be effectively neuro-attenuated by deletingthe γ₁34.5 neuropathogenesis genes. Chou et. al., Science (1990)250:1262-1266. The cellular interferon-induced eIF2α kinase PKR, a majorinnate host defense component, phosphorylates the critical host celltranslation initiation factor eIF2α in response to viral infection.Phosphorylated eIF2α blocks translation initiation thereby precludingthe manufacturing of viral polypeptides and progeny. The γ₁34.5 geneencodes a regulatory subunit of the cellular protein phosphatase 1 anddirects dephosphorylation of eIF2α which results in the production ofviral proteins and progeny. Chou et al., Proc. Natl. Acad. Sci. USA(1995) 92:10516-10520; He et. al., Proc. Natl. Acad. Sci. USA (1997)94:843-848. While γ₁34.5-deficient (Δ34.5) viruses are sufficientlyattenuated and safe (see, U.S. Pat. No. 7,981,669 by Coffin et al.),their anti-tumor efficacy in animal models is severely limited by theirconstrained ability to replicate in many types of cancer cells.

Failure of these Δ34.5 strains to propagate an infection throughout thetumor mass allows the cancer to simply regrow. See Mohr, Oncogene (2005)24:7697-7709. The HSV-1 Us11 gene has been shown to encode a functionexpressed very late in the viral growth cycle that antagonizes PKR andinnate host defenses. Viruses engineered to express Us11 very earlyfollowing infection (termed “immediate-early” of “IE”) allow Δ34.5mutant viruses to grow efficiently. Remarkably, Δ34.5 viruses thatexpress IE Us11 (Δ34.5 IE Us11) remain just as neuro-attenuated as theparental Δ34.5 strains, yet they replicate in and efficiently destroycancer cells, making them ideal oncolytic virus candidates. Mohr et.al., J. Virol. (2001) 75:5189-5196. In studies using independentlyconstructed viruses in different tumor models, engineering a Δ34.5mutant derivative to express IE Us11 resulted in a dramatic improvementin the ability of the virus to inhibit tumor growth. Taneja et. al.,Proc. Natl. Acad. Sci. USA (2001) 98:8804-8808; Todo et. al., Proc.Natl. Acad. Sci. USA (2001) 98:6396-6401; and Liu et al., Gene Therapy(2003) 1):292-303.

However, the above-described Δ34.5 IEUs11 oncolytic strains have a majordrawback, as engineering IE Us11 expression inactivates the neighboringUs12 gene, which encodes an important immunomodulatory polypeptide,ICP47, involved in blocking antigen presentation by inhibiting thetransporter associated with antigen presentation (TAP) 1/2. Mohr et al.,J. Virol. (1996) 75:5189-5196; Todo et al., Proc. Natl. Acad. Sci. USA(2001)98:6396-6401; Liu et al., Gene Therapy (2003) 10:292-303. Sincethe Us12 gene product acts to inhibit antigen presentation, its absenceresults in increased clearance of infected cells by the acquired immuneresponse. Goldsmith et al., J. Exp. Med. (1998) 187:341-348. Thus, Us12is likely required to ensure that the HSV-1 oncolytic virus is notprematurely cleared before it has a chance to spread through the tumortissue and complete its task of tumor eradication. This is especiallyimportant given the prevalence of HSV-1 and HSV-1-specific immunity(e.g., seropositivity) in the general population. Indeed, recentlypublished studies indicate that evasion of CD8+ T cells is critical forsuperinfection by a herpesvirus. Hansen et al., Science (2010)328:102-106. Although it is understood that Us12 prevents cytolyticT-cell recognition of infected cancer cells, it does not interfere withpresentation of tumor antigens on the surface of uninfected cells or,after infection begins, down-regulate existing cell surface complexesdisplaying tumor antigens. Hence, expression of Us12 immunomodulatoryactivity enhances viral spread and oncolysis but does not diminish theoverall immune response and/or potential for creating a tumorvaccination effect.

Δ34.5 IEUs11 HSV variants having intact Us12 were described in U.S. Pat.No. 7,731,952 by Mohr et al. While those Δ34.5 IEUs11 HSV variantsexpressed Us12, it is not possible to test those variants in murinemodels of, e.g., cancer, using immune-competent mice, because Us12cannot inhibit murine TAP, leading to the premature clearance ofvirus-infected cells, as discussed above.

Animal models are often instructive in understanding human diseases, andit would be useful to be able to test Δ34.5 IEUs11 HSV variants in suchmodels, especially ones that use immune-competent mice in order to moreclosely represent human diseases, such as cancer, in which most patientsare immune-competent and may also have anti-HSV specific memory T cells.Hence, there remains a need in the art for oncolytic viruses that evadeCD8+ T cells and/or avoid premature clearance by the immune system,particularly ones that can be tested in immune-competent murine andhuman models.

SUMMARY OF THE INVENTION

As discussed above, there remains a need in the art for variant HSV withimproved anti-tumor activity, including improved viral spreading andability to evade host immune responses, e.g., CD8+ cytolytic Tcell-mediated clearance of virally infected cells, that can be tested inimmune-competent murine models of disease, such as cancer.

Thus, a variant herpes simplex virus (HSV) having an intact endogenousUs12 encoding gene and an intact endogenous Us11 encoding gene, lackingfunctional ICP34.5 encoding genes, wherein each ICP34.5 encoding gene isreplaced by a polynucleotide cassette comprising: (a) a Us11 encodinggene operably associated with an immediate early (IE) promoter; and (b)at least one heterologous gene encoding a polypeptide capable ofenhancing an anti-tumor response is provided.

A heterologous gene can encode an immunomodulatory polypeptide, such asone selected from the group consisting of a TAP 1/2 (“TAP”) inhibitor,granulocyte macrophage colony stimulating factor (GM-CSF), tumornecrosis factor (TNF)-alpha and CD40 ligand (CD40L). Other non-limitingexamples of immunomodulatory polypeptides include for example, IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, G-CSF, IFN-α, IFN-γ, IL-20(MDA-7), and costimulator molecules such as B7-1 (CD80) and B7-2 (CD86).

The heterologous gene can also encode a prodrug converting enzyme. Theheterologous gene can also encode an enzyme that degrades or modifiesextra-cellular matrix components in order to facilitate viral spreadthrough the tumor, for example, a matrix metalloproteinase.

A variant HSV having an intact endogenous Us12 encoding gene and anintact endogenous Us11 encoding gene, lacking functional ICP34.5encoding genes, wherein each ICP34.5 encoding gene is replaced by apolynucleotide cassette comprising: (a) a Us11 encoding gene operablyassociated with an immediate early (IE) promoter; and (b) at least twoheterologous genes encoding a polypeptide capable of enhancing ananti-tumor response is also provided. The at least two heterologousgenes can, for example, encode a TAP inhibitor and a mammalian GM-CSF.The TAP inhibitor can inhibit a non-human TAP, such as, for example, amurine TAP. Preferably, the TAP inhibitor is the UL49.5 polypeptide frombovine herpesvirus. The at least two heterologous genes can also encode,for example, a TAP inhibitor and a prodrug converting enzyme. Theheterologous gene can also encode an enzyme that degrades or modifiesextra-cellular matrix components in order to facilitate viral spreadthrough the tumor, for example, a matrix metalloproteinase.

A variant herpes simplex virus (HSV) having an intact endogenous Us12encoding gene and an intact endogenous Us11 encoding gene, lackingfunctional ICP34.5 encoding genes, wherein each ICP34.5 encoding gene isreplaced by a polynucleotide cassette comprising: (a) a Us11 encodinggene operably associated with an immediate early (IE) promoter, and (b)a gene encoding an inhibitor of antigen presentation on class I majorhistocompatibility complex (MHC) molecules, wherein said inhibitor iscapable of inhibiting antigen presentation on the surface of virallyinfected tumor cells is also provided. Preferred inhibitors of antigenpresentation are TAP inhibitors.

A variant herpes simplex virus (HSV) having an intact endogenous Us12encoding gene and an intact endogenous Us11 encoding gene, lackingfunctional ICP34.5 encoding genes, wherein each ICP34.5 encoding gene isreplaced by a polynucleotide cassette comprising: (a) a Us11 encodinggene operably associated with an immediate early (IE) promoter; (b) agene encoding an inhibitor of antigen presentation on class I majorhistocompatibility complex (MHC) molecules, wherein said inhibitor iscapable of inhibiting antigen presentation on the surface of virallyinfected tumor cells is also provided; and (c) a heterologous geneencoding a polypeptide capable of enhancing an anti-tumor response isalso provided. Preferably, the heterologous gene encodes GM-CSF.Preferably, the IE promoter is an α27 IE promoter. In some embodiments,the heterologous gene is operably associated with a promoter selectedfrom the group consisting of a CMV promoter and an EF1α promoter.Preferably, the TAP inhibitor is a bovine herpesvirus (BHV) UL49.5polypeptide.

A variant herpes simplex virus (HSV) having an intact endogenous Us12encoding gene and an intact endogenous Us11 encoding gene, lackingfunctional ICP34.5 encoding genes, wherein each ICP34.5 encoding gene isreplaced with a polynucleotide cassette comprising a nucleic acidsequence set forth in one of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24 and SEQ ID NO: 25 is also provided.

A variant herpes simplex virus (HSV) having a genome sequence set forthin SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ IDNO: 30 is also provided.

A pharmaceutical formulation comprising a variant HSV of the inventionand a pharmaceutically acceptable carrier for administration to tumorcells is provided herein. Preferably, a pharmaceutical formulationcomprises a variant herpes simplex virus (HSV) having an intactendogenous Us12 encoding gene and an intact endogenous Us11 encodinggene, lacking functional ICP34.5 encoding genes, wherein each ICP34.5encoding gene is replaced by a polynucleotide cassette comprising: (a) aUs11 encoding gene operably associated with an immediate early (IE)promoter; and (b) at least one heterologous gene encoding a polypeptidecapable of enhancing an anti-tumor response, and a pharmaceuticallyacceptable carrier for administration to tumor cells.

A pharmaceutical formulation can also comprise a variant HSV having anintact endogenous Us12 encoding gene and an intact endogenous Us11encoding gene, lacking functional ICP34.5 encoding genes, wherein eachICP34.5 encoding gene is replaced by a polynucleotide cassettecomprising: (a) a Us11 encoding gene operably associated with animmediate early (IE) promoter; and (b) at least two heterologous genesencoding a polypeptide capable of enhancing an anti-tumor response, anda pharmaceutically acceptable carrier for administration to tumor cells.

A pharmaceutical formulation can also comprise a variant herpes simplexvirus (HSV) having an intact endogenous Us12 encoding gene and an intactendogenous Us11 encoding gene, lacking functional ICP34.5 encodinggenes, wherein each ICP34.5 encoding gene is replaced by apolynucleotide cassette comprising: (a) a Us11 encoding gene operablyassociated with an immediate early (IE) promoter; and (b) a geneencoding an inhibitor of antigen presentation on class I majorhistocompatibility complex (MHC) molecules, wherein said inhibitor iscapable of inhibiting antigen presentation on the surface of virallyinfected tumor cells and, optionally, (c) a heterologous gene encoding apolypeptide capable of enhancing an anti-tumor response, and apharmaceutically acceptable carrier for administration to tumor cells.

A pharmaceutical formulation can also comprise a variant herpes simplexvirus (HSV) having an intact endogenous Us12 encoding gene and an intactendogenous Us11 encoding gene, lacking functional ICP34.5 encodinggenes, wherein each ICP34.5 encoding gene is replaced with apolynucleotide cassette comprising a nucleic acid sequence set forth inone of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 andSEQ ID NO: 25.

A pharmaceutical formulation can also comprise a variant herpes simplexvirus (HSV) having a genome sequence set forth in SEQ ID NO: 26, SEQ IDNO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30.

Preferably, a pharmaceutical formulation provided herein is foradministration to tumor cells in situ. In some embodiments, thepharmaceutical formulation comprises a variant HSV that selectivelyinfects bladder cancer cells, human melanoma cells, human ovarian cancercells, or human glioblastoma cells.

A method for killing tumor cells in a subject comprising: administeringto a subject in need thereof a pharmaceutical formulation describedabove under conditions effective to kill tumor cells in the subject isalso provided. Non-limiting examples of tumor cells that can be killedaccording to the methods described herein, include, e.g., astrocytoma,oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma,Schwannoma, neurofibrosarcoma, medulloblastoma, melanoma cells,pancreatic cancer cells, prostate carcinoma cells, breast cancer cells,lung cancer cells, colon cancer cells, hepatoma cells, mesothelioma,bladder cancer cells, and epidermoid carcinoma cells. In certainembodiments, the virus can selectively replicate in human bladder cancercells or human melanoma cells. Administration to a subject can becarried out by injection, infusion, instillation or inhalation. In anyof the above embodiments, a subject can be a mammal, such as a human.

In one embodiment, a method for treating cancer is also provided,wherein the method comprises administering to an individual in need oftreatment, a therapeutically effective amount of a pharmaceuticalformulation described above. In certain embodiments, the cancer isselected from the group consisting of bladder cancer, melanoma, ovariancancer and glioblastoma.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-IC illustrate genetic properties of a wild-type HSV-1 (FIG. 1A)and of modified HSV-1 OncoVEX^(GMCSF) where GM-CSF is under the controlof the Cytomegalovirus (CMV) promoter (FIG. 1B) and OV-2711, expressingUs11 fused to an immediate early (IE) promoter. (FIG. 1C). Boxed regionsdesignate inverted terminal repeat (TR) regions that flank the uniqueshort (U_(s)) and unique long (U_(L)) components, represented by solidlines. Dotted lines indicate an expanded view of a region of the genome.The U_(s)-TRs junction region containing the Us11 and Us12 open readingframes (ORFs), designated by open rectangles, appears expanded. Starsrepresent the respective cis-acting promoter elements, where star-11indicates the promoter for Us11 and star-12 indicates the promoter forUs12. The arrow above each box extending from the promoter elementdenotes the mRNA transcript that encodes each gene product. All of thesemRNAs are polyadenylated at a common polyadenylation signal (notdepicted) downstream from Us11. The Us12 mRNA is spliced, as indicatedby the dip in the arrow joining two non-contiguous regions to form themRNA.

FIG. 2 illustrates the γ34.5 locus-targeting vector constructionstrategy, FIGS. 3A-3D illustrate 22 constructs that can be made usingthe strategy shown in FIG. 2, and FIG. 4 illustrates the geneticconstructs used to generate five specific OV-2711 variant oncolyticviruses (OV) (OV-UL49.5, OV-UL49.5-fs, OV-mGM-CSF, OV-UL49.5/GM-CSF, andOV-UL49.5-fs/mGM-CSF). In these Figures, the γ34.5 locus-targetingvector, shown at the top of FIG. 2, FIG. 3A, and FIG. 4, is derived fromthe viral BamSP fragment. In each targeting vector, γ34.5, locatedbetween the DraI (specifically nt#125989 of X14112) and SacI(specifically nt#125065 of X14112) sites of BamSP, is replaced by theα27-Us11 dominant selectable marker. In this process, the SacI site isdestroyed and the DraI site is replaced by a PacI site. Below thetargeting vector, CMV and EF1α promoter cassettes expressing eitherUL49.5 (“49.5”) or GM-CSF (“GM”) flanked by the indicated restrictionendonuclease sites (marked by vertical arrows) are shown. CMV-basedcassettes are terminated by the BGH polyadenylation signal (filledcircle) and EF1α terminated by the SV40 late polyadenylation signal(open square). FIG. 3A, at the top, shows the location of the gK genewithin the α27-promoter, and the proposed, but currentlyuncharacterized, location of the gK promoter (indicated by a star and“?”). In FIG. 4, the diamond in the UL49.5-fs open reading frame (ORF)is a single C nucleotide insertion between the second and third codonsof UL49.5 to create a frameshift (fs) mutation.

FIG. 5 is a flow diagram illustrating the strategy used to construct thetargeting vectors used to make recombinant oncolytic viruses, OV-UL49.5,OV-UL49.5-fs, OV-mGM-CSF, and OV-UL49.5/mGM-CSF. Open boxes in thediagram are constructs that were synthesized de novo by GenScriptCorporation (Piscataway, N.J.). The filled boxes are the constructsderived from restriction enzyme cloning.

FIG. 6 contains a Southern blot result showing the presence of theindicated constructs in viral DNA from high titer viral stocks ofrecombinant HSV1 variants. Lanes 1 through 6, from left to right, showthe presence of the constructs for the following recombinant viruses(molecular size of fragment indicated in parentheses in base pairs(bp)), respectively: 1. OV-mGM-CSF (4130 bp); 2. OV-UL49.5/mGM-CSF (6020bp); 3. OV-UL49.5 (3995 bp); 4. OV-UL49.5-fs (3996 bp); 5. OV-2711 (2727bp); and 6. Δ34.5 (1085 bp).

FIG. 7A contains a Western blot result showing the expression of theUL49.5 polypeptide detected in Vero cells mock infected or infected withfive separate plaque purified isolates of OV-UL49.5/GM-CSF at amultiplicity of infection (MOI) equal to 1.

FIG. 7B contains a Western blot result showing the expression of UL49.5polypeptide in Vero cells infected with either wild-type (WT) Pattonstrain HSV-1 or OV-UL49.5 at a multiplicity of infection (MOI) equal to5.

FIGS. 8A and 8B are bar graphs quantifying the expression of mGM-CSFmRNA (FIG. 7A) and VP16 (FIG. 7B) as detected by qRT-PCR and normalizedto 18S rRNA signal in mouse Balb/c mammary 4T1 cancer cells mockinfected or infected with wild-type (WT) Patton strain HSV-1, or withOV-mGM-CSF or OV-UL49.5/GM-CSF.

FIG. 9 is a line graph quantifying the replication (expressed as plaqueforming units (pfu)/ml) of the indicated viruses (wild-type (WT),Δ34.5ΔICP47, OV-2711 and Δ34.5) in infected MBT-2 cell monolayers overtime (hours post-infection (PI)).

DETAILED DESCRIPTION

Overview

The present invention provides novel variant herpes simplex viruses(HSV) with improved anti-tumor activity and improved ability to evadehost immune responses. In particular, the variant HSV provided hereinare non-neurovirulent, replicate in and destroy neoplastic cells, andhave improved activity in syngeneic, immune-competent murine models,e.g., for human bladder and other types of cancers.

Thus, in a preferred embodiment, a variant HSV of the invention has anintact Us12 encoding gene and/or an intact endogenous Us11 encodinggene, and lacks functional ICP34.5 encoding genes, wherein each ICP34.5encoding gene is replaced by a polynucleotide cassette comprising: (a) aUs11 encoding gene operably associated with an immediate early (TE)promoter; and (b) a gene encoding an inhibitor of antigen presentationon class I major histocompatibility (MHC) molecules (e.g., a TAPinhibitor) and/or a gene encoding a polypeptide capable of enhancing ananti-tumor response, such as GM-CSF, TNF-α, an interleukin (for exampleIL12), an interferon (such as IFN-γ) a chemokine such as RANTES or amacrophage inflammatory protein (MIP) (for example, MIP-3), or anotherimmunomodulatory molecule such as B7.1 (CD80), B7.2 (CD86) or CD40L, toname a few. In one preferred embodiment, the polypeptide is a mammalianGM-CSF. The heterologous gene can also encode an enzyme that degrades ormodifies extra-cellular matrix components in order to facilitate viralspread through the tumor, for example, a matrix metalloproteinase.

In another embodiment, a variant HSV of the invention has an intact Us12encoding gene and/or an intact endogenous Us11 encoding gene, and lacksfunctional ICP34.5 encoding genes, wherein each ICP34.5 encoding gene isreplaced by a polynucleotide cassette comprising at least one geneencoding a heterologous polypeptide. In a preferred embodiment, thevariant HSV has an intact Us12 encoding gene and/or an intact endogenousUs11 encoding gene, and lacks functional ICP34.5 encoding genes, whereineach ICP34.5 encoding gene is replaced by a polynucleotide cassettecomprising at least two genes encoding heterologous polypeptides. Incertain embodiments, a heterologous polypeptide is selected from aninhibitor of antigen presentation on class I major histocompatibility(MHC) molecules (e.g., a TAP inhibitor), a polypeptide capable ofenhancing an anti-tumor response (such as, but not limited to, GM-CSF,TNF-α, an interleukin (for example IL12), an interferon (such as IFN-γ)a chemokine (e.g., RANTES or a macrophage inflammatory protein (MIP)(e.g., MIP-3)), another immunomodulatory molecule (e.g., B7.1 (CD80),B7.2 (CD86), CD40L, etc.), and a prodrug converting enzyme. Theheterologous gene can also encode an enzyme that degrades or modifiesextra-cellular matrix components in order to facilitate viral spreadthrough the tumor, for example, a matrix metalloproteinase.

In a particularly preferred embodiment, a variant HSV of the inventionhas an intact endogenous Us12 encoding gene and an intact endogenousUs11 encoding gene, and lacks functional ICP34.5 encoding genes, whereineach ICP34.5 encoding gene is replaced by a polynucleotide cassettecomprising: a Us11 encoding gene operably associated with an immediateearly (IE) promoter, a gene encoding a mammalian GM-CSF, and a geneencoding a TAP inhibitor.

Although the invention is not limited by any particular theory ormechanism of action, the ability of the virus to inhibit TAP, e.g., viaICP47, the gene product of Us12, increases its ability to evade hostimmune responses (e.g., cytolytic CD8 T cell responses), therebyimproving the ability of the virus to spread throughout and kill tumorcells before being cleared by the host immune response. Furthermore, incertain embodiments, the variant HSV of the invention are particularlyuseful in animal models, e.g., rodent models of cancer, because theyadditionally comprise a gene encoding a TAP inhibitor active on murineTAP (e.g., UL49.5).

In certain embodiments, a variant HSV of the invention does notnecessarily have an intact endogenous Us12 gene. It is preferable,however, if the variant HSV does not have an intact endogenous Us12gene, that the variant HSV expresses a heterologous gene encoding apolypeptide having a substantially similar function (e.g., an immuneevasion function, such as a TAP inhibitor or other inhibitor of antigenpresentation on the virally infected cell surface by class I MHCmolecules) as that encoded by the Us12 gene.

Definitions

The following definitions are provided for clarity and illustrativepurposes only, and are not intended to limit the scope of the invention.

As used herein, the term “intact endogenous gene” in the context of avariant HSV of the invention refers to a gene (e.g., Us11 or Us12) thatis a naturally occurring gene in its naturally occurring location in theHSV genome. Intact endogenous genes may be fused to a heterologous gene.For example, endogenous Us11 may be fused to GFP, but as long as Us11 isfound in its naturally occurring location in the HSV genome, it is stillan intact endogenous gene within the meaning of the term as used herein.

As used herein, the phrase “lacking functional ICP34.5 encoding genes”in the context of a variant HSV of the invention means that each of thetwo genes encoding ICP34.5 in the HSV genome have been partially orcompletely deleted, replaced, rearranged, or otherwise altered such thatfunctional ICP34.5 polypeptide is not expressed by the HSV. Similarly,replacement of the ICP34.5 encoding gene (e.g., in the phrase “eachICP34.5 encoding gene is replaced”) means that a heterologous sequence,e.g., in a gene expression cassette, is substituted for all or part ofthe ICP34.5 encoding gene (γ₁34.5), e.g., by homologous recombination,such that functional ICP34.5 cannot be expressed from that gene. TheICP34.5 encoding gene may be replaced with any suitable heterologoussequence. That heterologous sequence may subsequently be replaced withanother heterologous sequence. For example, as described in Example 2,below, the ICP34.5 encoding gene was first replaced byBeta-glucoronidase to delete the ICP34.5 encoding gene, and thenB-glucoronidase was replaced with IE-Us11.

The terms “polynucleotide cassette” and “gene expression cassette” meansa manipulable fragment of DNA carrying, and capable of expressing, oneor more genes of interest between one or more sets of restriction sites.It can be transferred from one DNA sequence (usually on a vector) toanother by ‘cutting’ the fragment out using restriction enzymes and‘pasting’ it back into the new context. Typically, the DNA fragment(nucleic acid sequence) is operatively associated with expressioncontrol sequence elements which provide for the proper transcription andtranslation of the target nucleic acid sequence(s) (genes). Suchsequence elements may include a promoter and a polyadenylation signal.The “polynucleotide cassette” may further comprise “vector sequences.”By “vector sequences” is meant any of several nucleic acid sequencesestablished in the art which have utility in the recombinant DNAtechnologies of the invention to facilitate the cloning and propagationof the polynucleotide cassette including (but not limited to) plasmids,cosmids, bacterial artificial chromosomes, phage vectors, viral vectors,and yeast artificial chromosomes.

The term “heterologous” refers to a combination of elements notnaturally occurring. Thus, for example, a “heterologous gene” refers toa gene to be introduced to the genome of a virus, wherein that gene isnot normally found in the virus' genome or is a homolog of a geneexpressed in the virus from a different species (e.g., the bovine herpesvirus UL49.5 gene, which encodes for a TAP-inhibitor, is heterologouswhen inserted into the HSV genome, even though HSV also expresses a geneencoding a TAP-inhibitor (Us12), which has a different nucleic acidsequence and acts via a different biochemical mechanism.

Variant HSV of the invention infect and replicate in tumor cells,subsequently killing the tumor cells. Thus, such viruses are replicationcompetent. Preferably, they are selectively replication competent, i.e.,“selectively replicate” in tumor cells. This means that either theyreplicate in tumor cells and not in non-tumor cells, or that theyreplicate more effectively in tumor cells than in non-tumor cells. Forexample, where the variant HSV is used for treating a bladder tumor, thevariant HSV is capable of replicating in the bladder tumor cells but notin the surrounding tissue. Cells in which the virus is able to replicateare permissive cells. Measurement of selective replication competencecan be carried out by the tests described herein for measurement ofreplication and tumor cell-killing capacity, and also analyzed by thestatistical techniques mentioned herein if desired.

The phrase “enhancing an anti-tumor response” in the context of avariant HSV mean that the “anti-tumor” response induced followinginfection with a variant HSV, as measured, for example, and withoutlimitation, by decreased tumor growth, decreased tumor metastases,increased tumor cell death, increased CD8 T cell tumor infiltration,increased CD8 T cell-mediated tumor cell killing, increased levels ofanti-tumor immune cells in the animal or human, and/or increasedinduction of anti-tumor immunity, is greater compared to the anti-tumorresponse in the control, e.g., in tumor cells following infection with,e.g., a Δ34.5 HSV lacking intact endogenous Us12 gene. By way ofexample, and without limitation, an anti-tumor response is enhanced by avariant HSV if the variant HSV increases tumor cell death by, e.g., atleast 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, atleast 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, atleast 45-fold, at least 50-fold, at least 100-fold, at least 200-fold,at least 500-fold, at least 1000-fold or more, compared to the control.

As used herein, an “immunomodulatory polypeptide” in the context of avariant HSV of the invention refers to a polypeptide that is capable ofaltering the immune response to either the variant HSV or the host cell(i.e., the cell infected by the variant HSV), and/or to an uninfectedhost tumor cell. For example, one immunomodulatory polypeptideencompassed by the term is a TAP inhibitor polypeptide, such as, but notlimited to, UL49.5 polypeptide from bovine herpes virus (BHV). While notintending to be bound by theory or by one particular mechanism ofaction, TAP inhibitor polypeptides are thought to prevent presentationof viral antigens on the host cell's MHC molecules, thereby preventingrecognition of virally-infected cells by the host's immune system (e.g.,by cytolytic CD8 T cells). Thus, TAP inhibitors downmodulate the hostimmune response's ability to identify and kill virally infected cells.Other immunomodulatory polypeptides, however, include immunostimulatorypolypeptides, such as, but not limited to, GM-CSF, TNF-α and CD40L.Those exemplary polypeptides recruit and/or activate immune cells toinfiltrate tumors, process immunoactive molecules, recognize tumor cellsand/or lyse tumor cells (e.g., help mediate the oncolytic function ofthe variant HSV of the invention), and, therefore, upmodulate the hostimmune response. Importantly, in certain embodiments, the presence ofimmunostimulatory polypeptides, e.g., GM-CSF, which can enhance immunerecruitment to virally infected cells and tumors, has the potential tobe deleterious to viral infection of tumor cells and viral spreadthroughout tumor cells. Thus, it is particularly preferred that thevariant HSV of the invention additionally comprise a heterologouspolypeptide that is capable of enhancing the immune evasioncapabilities, and therefore the replication and spread, of the variantHSV, such as, but not limited to polypeptides that inhibit viral antigenpresentation by infected cells (e.g., UL49.5 from BHV).

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a polypeptide encoded by the gene or DNA sequence. As usedherein, a gene or DNA sequence is expressed in or by a virus to form an“expression product” such as a polypeptide. The expression productitself, e.g., the resulting polypeptide, may also be said to be“expressed” by the virus.

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of amino acidswhich comprise all or part of one or more polypeptides (e.g., proteins),and may or may not include regulatory DNA sequences, such as promotersequences, which determine for example the conditions under which thegene is expressed. Some genes, which are not structural genes, may betranscribed from DNA to RNA, but are not translated into an amino acidsequence. Other genes may function as regulators of structural genes oras regulators of DNA transcription.

A coding sequence is “under the control of” or “operatively associatedwith” expression control sequences in a virus or cell when RNApolymerase transcribes the coding sequence into RNA, particularly mRNA,which is then spliced (if it contains introns) and translated into thepolypeptide encoded by the coding sequence.

The term “expression control sequence” refers to a promoter and anyenhancer or suppression elements that combine to regulate thetranscription of a coding sequence. In a preferred embodiment, theelement is a transcriptional promoter.

A sequence “encoding” an expression product, such as a polypeptide, is aminimum nucleotide sequence that, when expressed, results in theproduction of that polypeptide.

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown, infected or used or manipulated in any wayfor the production of a substance by the cell or to grow, test, screen,or carry out another desired activity on, a variant HSV of theinvention. For example, a host cell may be one that is manipulated toexpress a particular gene, a DNA or RNA sequence, a polypeptide. In apreferred embodiment, a host cell is any one which is capable of beinginfected with a variant HSV (or control HSV) of the invention, e.g., forscreening or other assays that are described infra, e.g., for screeningthe activity, replication and protein synthesis efficiency of variantHSV of the invention. Such suitable cells are well known in the art.Host cells may be cultured in vitro or one or more cells in a non-humananimal (e.g., a transgenic animal or a transiently transfected animal).Exemplary suitable host cells include, but are not limited to, UMUC3,T24, J82 and EJ (MGH-UI), J82 (COT), RT4, RT112, TCCSuP and SCaBERcells.

“Treating” or “treatment” of a state, disorder or condition includes:(1) preventing or delaying the appearance of clinical or sub-clinicalsymptoms of the state, disorder or condition developing in a mammal thatmay be afflicted with or predisposed to the state, disorder or conditionbut does not yet experience or display clinical or subclinical symptomsof the state, disorder or condition; or (2) inhibiting the state,disorder or condition, i.e., arresting, reducing or delaying thedevelopment of the disease or a relapse thereof (in case of maintenancetreatment) or at least one clinical or sub-clinical symptom thereof; or(3) relieving the disease, i.e., causing regression of the state,disorder or condition or at least one of its clinical or sub-clinicalsymptoms.

For example, in relation to cancer, the term “treat” may mean to relieveor alleviate at least one symptom selected from the group consisting oftumor growth, metastasis, sensitivity of tumor cells to treatments suchas chemotherapy, radiation therapy, thermotherapy, etc. The term “treat”also denotes to arrest, delay the onset (i.e., the period prior toclinical manifestation of a disease) and/or reduce the risk ofdeveloping or worsening a disease. In a specific embodiment, treatingcancer comprises killing a tumor cell, e.g., with an oncolytic virus ofthe invention.

The benefit to a subject to be treated is either statisticallysignificant or at least perceptible to the patient or to the physician.

“Patient” or “subject” refers to mammals, for example and withoutlimitation, rodents (e.g., mice and rats), dogs, cats, cows, sheep,primates, and includes human and veterinary subjects.

An “effective amount” of a compound of the present invention includesdoses that partially or completely achieve the desired therapeutic,prophylactic, and/or biological effect. The actual amount effective fora particular application depends on the condition being treated and theroute of administration. The effective amount for use in humans can bedetermined from animal models.

As used herein the term “therapeutically effective” applied to dose oramount refers to that quantity of a compound or composition (e.g.,pharmaceutical composition) that is sufficient to result in a desiredactivity upon administration to an animal in need thereof. Thus, withinthe context of the present invention, the term “therapeuticallyeffective amount” refers to that quantity of a compound or compositionthat is sufficient to treat at least one symptom of a cancer, such asbut not limited to cancer cell proliferation, tumor growth, resistanceto apoptosis, and angiogenesis, and/or to inhibit metastasis of a cancercell. When a combination of active ingredients is administered, aneffective amount of the combination may or may not include amounts ofeach ingredient that would have been effective if administeredindividually. A “prophylactically effective amount” is an amount of apharmaceutical composition that, when administered to a subject, willhave the intended prophylactic effect, e.g., preventing or delaying theonset (or recurrence) of cancer, or reducing the likelihood of the onset(or recurrence) of cancer or cancer symptoms. The full prophylacticeffect does not necessarily occur by administration of one dose, and mayoccur only after administration of a series of doses. Thus, aprophylactically effective amount may be administered in one or moreadministrations.

The term “about” or “approximately” means within an acceptable range forthe particular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,e.g., the limitations of the measurement system. For example, “about”can mean a range of up to 20%, preferably up to 10%, more preferably upto 5%, and more preferably still up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5 fold, and more preferably within 2 fold, of a value. Unlessotherwise stated, the term ‘about’ means within an acceptable errorrange for the particular value, such as ±1-20%, preferably ±1-10% andmore preferably ±1-5%.

As used herein, the terms “mutant” and “mutation” refer to anydetectable change in genetic material (e.g., DNA) or any process,mechanism, or result of such a change. This includes gene mutations, inwhich the structure (e.g., DNA sequence) of a gene is altered, any geneor DNA arising from any mutation process, and any expression product(e.g., polypeptide) expressed by a modified gene or DNA sequence. Asused herein, the term “mutating” refers to a process of creating amutant or mutation.

The term “nucleic acid hybridization” refers to anti-parallel hydrogenbonding between two single-stranded nucleic acids, in which A pairs withT (or U if an RNA nucleic acid) and C pairs with G. Nucleic acidmolecules are “hybridizable” to each other when at least one strand ofone nucleic acid molecule can form hydrogen bonds with the complementarybases of another nucleic acid molecule under defined stringencyconditions. Stringency of hybridization is determined, e.g., by (i) thetemperature at which hybridization and/or washing is performed, and (ii)the ionic strength and (iii) concentration of denaturants such asformamide of the hybridization and washing solutions, as well as otherparameters. Hybridization requires that the two strands containsubstantially complementary sequences. Depending on the stringency ofhybridization, however, some degree of mismatches may be tolerated.Under “low stringency” conditions, a greater percentage of mismatchesare tolerable (i.e., will not prevent formation of an anti-parallelhybrid). See Molecular Biology of the Cell, Alberts et al., 3rd ed., NewYork and London: Garland Publ., 1994, Ch. 7.

Typically, hybridization of two strands at high stringency requires thatthe sequences exhibit a high degree of complementarity over an extendedportion of their length. Examples of high stringency conditions include:hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at65° C., followed by washing in 0.1×SSC/0.1% SDS at 68° C. (where 1×SSCis 0.15M NaCl, 0.15M Na citrate) or for oligonucleotide moleculeswashing in 6×SSC/0.5% sodium pyrophosphate at about 37° C. (for 14nucleotide-long oligos), at about 48° C. (for about 17 nucleotide-longoligos), at about 55° C. (for 20 nucleotide-long oligos), and at about60° C. (for 23 nucleotide-long oligos)). Accordingly, the term “highstringency hybridization” refers to a combination of solvent andtemperature where two strands will pair to form a “hybrid” helix only iftheir nucleotide sequences are almost perfectly complementary (seeMolecular Biology of the Cell, Alberts et al., 3rd ed., New York andLondon: Garland Publ., 1994, Ch. 7).

Conditions of intermediate or moderate stringency (such as, for example,an aqueous solution of 2×SSC at 65° C.; alternatively, for example,hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at65° C., and washing in 0.2×SSC/0.1% SDS at 42° C.) and low stringency(such as, for example, an aqueous solution of 2×SSC at 55° C.), requirecorrespondingly less overall complementarity for hybridization to occurbetween two sequences. Specific temperature and salt conditions for anygiven stringency hybridization reaction depend on the concentration ofthe target DNA and length and base composition of the probe, and arenormally determined empirically in preliminary experiments, which areroutine (see Southern, J. Mol. Biol. 1975; 98: 503; Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 2, ch. 9.50, CSHLaboratory Press, 1989; Ausubel et al. (eds.), 1989, Current Protocolsin Molecular Biology, Vol. I, Green Publishing Associates, Inc., andJohn Wiley & Sons, Inc., New York, at p. 2.10.3).

As used herein, the term “standard hybridization conditions” refers tohybridization conditions that allow hybridization of sequences having atleast 75% sequence identity. According to a specific embodiment,hybridization conditions of higher stringency may be used to allowhybridization of only sequences having at least 80% sequence identity,at least 90% sequence identity, at least 95% sequence identity, or atleast 99% sequence identity.

Nucleic acid molecules that “hybridize” to any desired nucleic acids ofthe present invention may be of any length. In one embodiment, suchnucleic acid molecules are at least 10, at least 15, at least 20, atleast 30, at least 40, at least 50, and at least 70 nucleotides inlength. In another embodiment, nucleic acid molecules that hybridize areof about the same length as the particular desired nucleic acid.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear (e.g., restrictionfragments) or circular DNA molecules, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

As used herein, the term “homologs” refers to genes in different speciesthat apparently evolved from a common ancestral gene by speciation.Normally, homologs retain the same function through the course ofevolution. Identification of homologs can provide reliable prediction ofgene function in newly sequenced genomes. Sequence comparison algorithmsthat can be used to identify homologs include without limitation BLAST,FASTA, DNA Strider, and the GCG pileup program. Homologs often have highsequence similarity. The present invention encompasses all homologs ofthe desired polypeptide.

The terms “percent (%) sequence similarity”, “percent (%) sequenceidentity”, and the like, generally refer to the degree of identity orcorrespondence between different nucleotide sequences of nucleic acidmolecules or amino acid sequences of polypeptides that may or may notshare a common evolutionary origin (see Reeck et al., supra). Sequenceidentity can be determined using any of a number of publicly availablesequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.), etc.

To determine the percent identity between two amino acid sequences ortwo nucleic acid molecules, the sequences are aligned for optimalcomparison purposes. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., percent identity=number of identical positions/total number ofpositions (e.g., overlapping positions)×100). In one embodiment, the twosequences are, or are about, of the same length. The percent identitybetween two sequences can be determined using techniques similar tothose described below, with or without allowing gaps. In calculatingpercent sequence identity, typically exact matches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1990,87:2264, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA1993, 90:5873-5877. Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul et al., J. Mol. Biol. 1990; 215: 403.BLAST nucleotide searches can be performed with the NBLAST program,score=100, wordlength=12, to obtain nucleotide sequences homologous tosequences of the invention. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3, to obtain amino acidsequences homologous to protein sequences of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., Nucleic Acids Res. 1997, 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationship between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov/BLAST/ on theWorldWideWeb. Another non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS 1988; 4: 11-17. Such an algorithm is incorporated intothe ALIGN program (version 2.0), which is part of the GCG sequencealignment software package. When utilizing the ALIGN program forcomparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used.

In a preferred embodiment, the percent identity between two amino acidsequences is determined using the algorithm of Needleman and Wunsch (J.Mol. Biol. 1970, 48:444-453), which has been incorporated into the GAPprogram in the GCG software package (Accelrys, Burlington, Mass.;available at accelrys.com on the WorldWideWeb), using either a Blossum62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or4, and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in the GCG software package using aNWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and alength weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set ofparameters (and the one that can be used if the practitioner isuncertain about what parameters should be applied to determine if amolecule is a sequence identity or homology limitation of the invention)is using a Blossum 62 scoring matrix with a gap open penalty of 12, agap extend penalty of 4, and a frameshift gap penalty of 5.

Statistical analysis of the properties described herein may be carriedout by standard tests, for example, t-tests, ANOVA, or Chi squaredtests. Typically, statistical significance will be measured to a levelof p=0.05 (5%), more preferably p=0.01, p=0.001, p=0.0001, p=000001.

Structure of Herpes Simplex Viruses and Variants

The variant HSV of the invention may be derived from a herpes simplexvirus (HSV) strain. The HSV strain may be an HSV-1 or HSV-2 strain, or aderivative thereof, and is preferably HSV-1. For example, a variant HSVof the invention may be derived from a wild-type HSV-1, strain 17,having GenBank Accession No. X14112 and the nucleic acid sequence setforth in SEQ ID NO: 1 in the sequence listing.

HSV strains of the invention may be “laboratory” or “non-laboratory”(“clinical”) strains. Laboratory strains in current use include HSV-1strain F, HSV-1 strain 17, HSV-1 strain KOS, and strain Patton. Clinicalstrains useful in the invention typically have improved oncolyticactivity compared to HSV-1 strains F, 17+ and KOS strains withequivalent modifications.

While the sequence for the complete genome of strain 17 of HSV-1 isprovided herein as an example, the nucleic acid sequence of any suitablelab strain (e.g., F, KOS, and Patton) and/or clinical isolate can beused according to the present invention. Derivatives of HSV, which mayalso be used according to the invention described herein, include butare not limited to inter-type recombinants containing DNA from HSV-1 andHSV-2 strains. Such inter-type recombinants are described in the art,for example in Thompson et al., “DNA sequence and RNA transcriptionthrough a site of recombination in a non-neurovirulent herpes simplexvirus intertypic recombinant,” Virus Genes, 1(3): 275-286, 1998; andMeignier et al., “In vivo behaviour of genetically engineered herpessimplex viruses R7017 and R7020: construction and evaluation inrodents,” J. Infect. Dis., 158(3): 602-614, 1988. Derivatives preferablyhave at least 70% sequence homology to either the HSV-1 or HSV-2 genome,more preferably at least 80%, even more preferably at least 90 or 95%.More preferably, a derivative has at least 70% sequence identity toeither the HSV-1 or HSV-2 genome, more preferably at least 80% identity,even more preferably at least 90%, 95% or 98% identity.

HSV-1 is a double-stranded DNA virus, having a genome size of 152 kb,which is replicated and transcribed in the nucleus of a host cell. HSV-1has two unique genome segments: Unique-long (U_(L)) and Unique-short(U_(s)). As shown in FIG. 1A, both unique sequences are flanked byinverted terminal repeats. In wild-type HSV-1, the γ₁34.5 gene, whichencodes ICP34.5 polypeptide and confers neurovirulence [see, Chou J, etal. “Mapping of herpes simplex virus-1 neurovirulence to gamma 1 34.5, agene nonessential for growth in culture.” Science 1990; 250:1262-6], isa diploid element located within the inverted repeats flanking U_(L).The Us12 gene, located in the U_(s) segment, is expressed very earlyduring infection by an immediate early promoter. The Us11 gene, a γ₂gene, is expressed late in viral infection by a separate promotercontained within the Us12 gene.

In the HSV-1 strain 17 genome (GenBank Accession No. X14112) (SEQ ID NO:1), the ORF for Us11 is found at nucleotides 144761-145246, and has thefollowing sequence:

(SEQ ID NO: 2) ctatacagacccgcgagccgtacgtggttcgcggggggtgcgtggggtccggggctcccggggagaccggggctcccggggagaccggggctccctgggagaccggggttgtcgtggatccctggggtcacgcggtaccctggggtctctgggagctcgcggtactctgggttccctaggttctcggggtggtcgcggaacccggggctcccggggaacacgcggtgtcctggggattgttggcggtcggacggcttcagatggcttcgagatcgtagtgtccgcaccgactcgtagtagacccgaatctccacattgccccgccgcttgatcattatcaccccgttgcgggggtccggagatcatgcgcgggtgtcctcgaggtgcgtgaacacctctggggtgcatgccggcggacggcacgccttttaagtaaacatctgggtcgcccggcccaactggggccgggggttgggtaggctcat.

In the HSV-1 strain 17 genome (GenBank Accession No. X14112) (SEQ ID NO:1), the ORF for Us12 is found at nucleotides 145311-145577, and has thefollowing sequence:

(SEQ ID NO: 3) tcaacgggttaccggattacggggactgtcggtcacggtcccgccggttcttcgatgtgccacacccaaggatgcgttgggggcgatttcgggcagcagcccgggagagcgcagcaggggacgctccgggtcgtgcacggcggttctggccgcctcccggtcctcacgcccccttttattgatctcatcgcgtacgtcggcgtacgtcctgggcccaacccgcatgagtccaggaaggtgtccgccatac cagggcccacgacat.

Exemplary sequences of certain genes encoded in the HSV-1 genome as wellan exemplary sequence of an entire HSV-1 genome are provided herein.However, it is to be understood that the present invention is notlimited to the exemplary sequences provided herein, and the inventionincludes variants of those sequences that encode the same gene(s), aswell as nucleic acid (gene) sequences encoding functional homologs(i.e., a polypeptide having substantially the same activity, but encodedby a different gene.

Multiple herpes simplex virus type 1 functions control translation byregulating phosphorylation of the initiation factor eIF2 on its alphasubunit. Both of the two known regulators, the γ₁34.5-encoded and Us11gene products, are produced late in the viral life cycle, although theγ₁34.5 gene is expressed prior to the γ₂ Us11 gene, as γ₂ genes requireviral DNA replication for their expression while γ₁ genes do not. TheICP34.5 polypeptide, the product of the γ₁34.5 gene, through aGADD34-related domain, binds a cellular phosphatase (PP1α), maintainingpools of active, unphosphorylated eIF2. Infection of a variety ofcultured cells with an ICP34.5 mutant virus results in the accumulationof phosphorylated eIF2α and the inhibition of translation prior to thecompletion of the viral lytic program. Ectopic, immediate-early Us11expression prevents cIF2α phosphorylation and the inhibition oftranslation observed in cells infected with a ICP34.5 mutant byinhibiting activation of the cellular kinase PKR and the subsequentphosphorylation of eIF2α. Further, the Us11 polypeptide is critical forproper late translation rates. The shutoff of protein synthesis observedin cells infected with an ICP34.5 mutant virus results from the combinedloss of ICP34.5 and Us11 functions, as the Us11 mRNA is not translatedin cells infected with an ICP34.5 mutant.

Viral regions altered for the purposes described above may be eithereliminated (completely or partly), or made non-functional, orsubstituted by other sequences, for example, and without limitation, bya gene encoding a prodrug converting enzyme, a gene encoding apolypeptide capable of causing cell to cell fusion, a gene encoding animmunomodulatory polypeptide, or a gene encoding a function thatmodifies the extracellular matrix.

A derivative may have the sequence of a HSV-1 or HSV-2 genome modifiedby nucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50 or100 substitutions. The HSV-1 or HSV-2 genome may alternatively oradditionally be modified by one or more insertions and/or deletionsand/or by an extension at either or both ends.

The properties of the variant HSV with respect to tumor cells can bemeasured in any manner known in the art. For example, the capacity of avariant HSV to infect a tumor cell can be quantified by measuring thevariant HSV's capacity to replicate in a tumor cell, as measured bygrowth measurements, e.g., by measuring virus growth (viral titer) incells over a period of 6, 12, 24, 36, 48 or 72 hours or longer. Asdescribed in the Examples, below, the ability of a variant HSV to infectand replicate within a tumor cell can be measured by determining thepercentage of cells exhibiting a cytopathic effect (cpe) followinginfection with the variant HSV, wherein a variant HSV having the abilityto infect cells will induce a cpe in at least about 50%, 60%, 70%, 80%or preferably 90% of the cells. The ability of a variant HSV to infectand replicate within a tumor cell may also be measured indirectly bymeasuring production of viral polypeptides (e.g., by ³⁵S cysteine andmethionine labeling followed by SDS-PAGE and autoradiography and Westernblot analysis).

The ability of a virus to kill tumor cells can be roughly quantitated byeye or more exactly quantitated by counting the number of live cellsthat remain over time for a given time point and multiplicity ofinfection (MOI) for given cell type. For example, comparisons may bemade over 24, 48 or 72 hours and using any known tumor cell type. Inparticular, UMUC3 invasive, high-grade bladder cancer, HT29 colorectaladenocarcinoma, LNCaP.FGC prostate adenocarcinoma, MDA-MB-231 breastadenocarcinoma, SK-MEL-28 malignant melanoma or U-87 MG glioblastomaastrocytoma cells can be used. Other examples of cell lines that arewell known in the art and which may be used include, but are not limitedto, HTB-161, SW620, A2780S, COLO205, A2780DDP, CX-1, SW948, SKBR3,MCF-7, HCT-15, CACO-2, A549, NEC, LX-1, T47D, B7474, DU145, PC3,SK-MEL-303, and LN-CAP cell lines. Any one of these cell types or anycombination of these cell types can be used, as may other tumor celltypes. It may be desirable to construct a standard panel of tumor celltypes for this purpose. To count the number of live cells remaining at agiven time point, the number of trypan blue-excluding cells (i.e., livecells) can be counted. Quantitation may also be carried out byfluorescence activated cell sorting (FACS) or MTT assay. Tumorcell-killing ability may also be measured in vivo, e.g., by measuringthe reduction in tumor volume engendered by a particular virus, asdescribed, e.g., in the Examples, below.

In order to determine the properties of variant HSV of the invention, itwill generally be desirable to use a standard laboratory referencestrain for comparison. Any suitable standard laboratory reference strainmay be used. In the case of HSV, it is preferred to use one or more ofHSV-1 strain 17+, HSV-1 strain F, HSV-1 strain KOS, or HSV-1 strainPatton. The reference strain will typically have equivalentmodifications to the strain of the invention being tested. Thus, thereference strain will typically have equivalent modifications, such asgene deletions and heterologous gene insertions. In the case of avariant HSV of the invention, where the ICP34.5 encoding genes have beenreplaced or otherwise rendered non-functional, the ICP34.5 encodinggenes will also have been rendered non-functional in the referencestrain. The modifications made to the reference strain may be identicalto those made to the strain of the invention. By this, it is meant thatthe gene disruptions in the reference strain will be in exactlyequivalent positions to those in the strain of the invention, e.g.,deletions will be of the same size and in the same place. Similarly, inthese embodiments, heterologous genes will be inserted in the sameplace, driven by the same promoter, etc. However, it is not essentialthat identical modifications be made. What is important is that thereference strain has functionally equivalent modifications, e.g., thatthe same genes are rendered non-functional and/or the same heterologousgene or genes is inserted.

Replacement of ICP34.5 Encoding Genes

The variant HSV of the invention have intact endogenous Us11 and Us12genes and the ICP34.5 encoding genes are replaced with a polynucleotidecassette comprising a Us11 gene operatively associated with an immediateearly (IE) promoter (“IE-Us11”). Preferably, the polynucleotide cassetteadditionally comprises one or more genes encoding a heterologouspolypeptide, as described herein.

By way of example, in the HSV-1 strain 17 genome (GenBank Accession No.X14112) (SEQ ID NO: 1), the ORF for the first ICP34.5 encoding (γ₁34.5)gene is found at nucleotides 513-1259, and has the following sequence:

(SEQ ID NO: 4) atggcccgccgccgccgccatcgcggcccccgccgcccccggccgcccgggcccacgggcgccgtcccaaccgcacagtcccaggtaacctccacgcccaactcggaacccgcggtcaggagcgcgcccgcggccgccccgccgccgccccccgccggtgggcccccgccttcttgttcgctgctgctgcgccagtggctccacgttcccgagtccgcgtccgacgacgacgatgacgacgactggccggacagccccccgcccgagccggcgccagaggcccggcccaccgccgccgccccccggccccggcccccaccgcccggcgtgggcccggggggcggggctgacccctcccaccccccctcgcgccccttccgccttccgccgcgcctcgccctccgcctgcgcgtcaccgcggagcacctggcgcgcctgcgcctgcgacgcgcgggcggggagggggcgccggagccccccgcgacccccgcgacccccgcgacccccgcgacccccgcgacccccgcgcgggtgcgcttctcgccccacgtccgggtgcgccacctggtggtctgggcctcggccgcccgcctggcgcgccgcggctcgtgggcccgcgagcgggccgaccgggctcggaccggcgccgggtggcggaggccgaggcggtcatcgggccgtgcctggggcccgaggcccgtgcccgggccctggcccgcggagccggcccggcgaactcggtctaa.

Further, the ORF for the second ICP34.5 encoding (γ₁34.5) gene is foundat nucleotides 125112-125858, and has the following sequence:

(SEQ ID NO: 5) ttagaccgagttcgccgggccggctccgcgggccagggcccgggcacgggcctcgggccccaggcacggcccgatgaccgcctcggcctccgccacccggcgccggaaccgagcccggtcggcccgctcgcgggcccacgagccgcggcgcgccaggcgggcggccgaggcccagaccaccaggtggcgcacccggacgtggggcgagaagcgcacccgcgcgggggtcgcgggggtcgcgggggtcgcgggggtcgcgggggtcgcggggggctccggcgccccctccccgcccgcgcgtcgcaggcgcaggcgcgccaggtgctccgcggtgacgcgcaggcggagggcgaggcgcggcggaaggcggaaggggcgcgagggggggtgggaggggtcagccccgccccccgggcccacgccgggcggtgggggccggggccggggggcggcggcggtgggccgggcctctggcgccggctcgggcggggggctgtccggccagtcgtcgtcatcgtcgtcgtcggacgcggactcgggaacgtggagccactggcgcagcagcagcgaacaagaaggcgggggcccaccggcggggggcggcggcggggcggccgcgggcgcgctcctgaccgcgggttccgagttgggcgtggaggttacctgggactgtgcggttgggacggcgcccgtgggcccgggcggccgggggcggcgggggccgcgatggcggcggcggcgggccat.

ICP34.5 encoding genes (γ₁34.5 genes) may be rendered functionallyinactive (e.g., replaced) by several techniques well known in the art.For example, they may be rendered functionally inactive by deletion(s),substitution(s) or insertion(s), preferably by deletion. Deletions mayremove one or more portions of the gene or the entire gene. For example,deletion of only one nucleotide may be made, resulting in a frame shift.However, preferably a larger deletion(s) is made, for example at least25%, more preferably at least 50% of the total coding and non-codingsequence (or alternatively, in absolute terms, at least 10 nucleotides,more preferably at least 100 nucleotides, most preferably, at least 1000nucleotides). It is particularly preferred to remove the entire gene andsome of the flanking sequences, e.g., by replacing the gene by inserting(e.g., via homologous recombination) one or more expression cassettescomprising heterologous gene(s) into the γ₁34.5 locus. Where two or morecopies of the gene are present in the viral genome it is preferred thatboth copies of the gene are rendered functionally inactive. HSV have twocopies of ICP34.5 encoding (γ₁34.5) genes, and thus, it is particularlypreferred that both copies of the ICP34.5 encoding genes are replacedand/or rendered functionally inactive.

In a preferred embodiment, both ICP34.5 encoding (γ₁34.5) genes of HSVare replaced with gene expression cassettes each comprising an IE-Us11gene and one or more heterologous polypeptides described herein.However, it is also possible that only one of the γ₁34.5 genes isreplaced with one or more gene expression cassettes comprising anIE-Us11 gene and one or more heterologous polypeptides, and the otherγ₁34.5 gene is deleted or otherwise rendered functionally inactivewithout inserting an expression cassette, or by inserting a differentgene expression cassette or combination of gene expression cassettes.

Non-limiting examples of the IE promoter operably linked to the Us11gene include α0, α4, α22, α27 and α47. In a preferred embodiment, the IEpromoter is α27. By way of example, the nucleic acid sequences encodingthe α0, α4, α22, α27 and α47 promoters in the HSV-1 strain 17 genome(GenBank Accession No. X14112) (SEQ ID NO: 1) are provided below:

The α0 promoter gene is diploid and found at nucleotides 1302-2166 and125069-124205, and has the following sequence:

(SEQ ID NO: 6) gagctccgcaccaagccgctctccggagagacgatggcaggagccgcgcatatatacgcttggagccagcccgccctcacagggcgggccgcctcgggggcgggactggccaatcggcggccgccagcgcggcggggcccggccaaccagcgtccgccgagtcttcggggcccggcccattgggcgggagttaccgcccaatgggccgggccgcccacttcccggtatggtaattaaaaacttgcaagaggccttgttccgcttcccggtatggtaattagaaactcattaatgggcggccccggccgccatcccgcttccggcaattcccgcggcccttaatgggcaaccccggtattccccgcctcccgcgccgcgcgtaaccactcccctggggttccgggttatgctaattgcttttttggcggaacacacggcccctcgcgcattggcccgcgggtcgctcaatgaacccgcattggtcccctggggttccgggtatggtaatgagtttcttcgggaaggcgggaagccccggggcaccgacgcaggccaagcccctgttgcgtcggcgggaggggcatgctaatggggttctttgggggacaccgggttgggcccccaaatcgggggccgggccgtgcatgctaatgatattattgggggcgccgggttggtccccggggacggggccgccccgcggtgggcctgcctcccctgggacgcgcggccattgggggaatcgtcactgccgcccctttggggaggggaaaggcgtggggtataagttagccctggcccgacagtctggtcgcatttgcacctcggcactcggagcgagacgcagcag ccaggcagactcg.

There are two α4 promoter genes composed of a shared portion of theterminal repeat short and either the unique short sequence containingthe α22 ORF or the ICP47 ORF: The α4 promoter gene with the unique shortsequence containing the α22 ORF is found at nucleotides 131399-136294,and has the following sequence:

(SEQ ID NO: 7) ggatccgtgtcggcagccgcgctccgtgtggacgatcggggcgtcctcgggctcatatagtcccaggggccggcgggaaggaggagcagcggaggccgccggccccccgcccccccggcgggcccaccccgaacggaattccattatgcacgaccccgccccgacgccggcacgccgggggcccgtggccgcggcccgttggtcgaacccccggccccgcccatccgcgccatctgccatgggcggggcgcgagggcgggtgggtccgcgccccgccccgcatggcatctcattaccgcccgatccggcggtttccgcttccgaccgcatgctaacgaggaacgggcagggggcggggcccgggccccgacttcccggttcggcggtaatgagatacgagccccgcgcgcccgttggccgtccccgggcaaaaaggtcccgcccgccggacgccgggaccaacgggacggcgggcggcccaagggccgcccgccttgccgcccccccattggccggcgggcgggaccgccccaagggggcggggccgccgggtaaaagaagtgagaacgcgaagcgttcgcacttcgtcccaatatatatatattattagggcgaagtgcgagcactggcgccgtgcccgactccgcgccggccccgggggcgggcccgggcggcggggggcgggtctctccggcgcacataaaggcccggcgcgaccgacgcccgcagacggcgccggccacgaacgacgggagcggctgcggagcacgcggaccgggagcgggagtcgcagagggccgtcggagcggacggcgtcggcatcgcgacgccccggctcgggatcgggatcgcatcggaaagggacacgcggacgcgggggggaaagacccgcccaccccacccacgaaacacaggggacgcaccccgggggcctccgacgacagaaacccaccggtccgccttattgcacgggtaagcaccttgggtgggcggaggagggggggacgcgggggcggaggaggggggacgcgggggcggaggaggggggacgcgggggcggaggaggggggacgcgggggcggaggaggggggacgcgggggcggaggagggggctcacccgcgttcgtgccttcccgcaggaggaacgtcctcgtcgaggcgaccggcggcgaccgttgcgtggaccgcttcctgctcgtcgggcggggggaagccactgtggtcctccgggacgttttctggatggccgacatttccccaggcgcttttgcgccttgtgtaaaagcgcggcgtcccgctctccgatccccgcccctgggcacgcgcaagcgcaagcgcccttcccgccccctctcatcggagtctgaggtagaatccgatacagccttggagtctgaggtcgaatccgagacagcatcggattcgaccgagtctggggaccaggatgaagccccccgcatcggtggccgtagggccccccggaggcttggggggcggttattctggacatgtcggcggcatccaccacggggacggaaacggatgcgtcggtgtcggacgaccccgacgacacgtccgactggtcttatgacgacattcccccacgacccaagcgggcccgggtaaacctgcggctcacgagctctcccgatcggcgggatggggttatttttcctaagatggggcgggtccggtctacccgggaaacgcagccccgggcccccaccccgtcggccccaagcccaaatgcaatgctacggcgctcggtgcgccaggcccagaggcggagcagcgcacgatggacccccgacctgggctacatgcgccagtgtatcaatcagctgtttcgggtcctgcgggtcgcccgggacccccacggcagtgccaaccgcctgcgccacctgatacgcgactgttacctgatgggatactgccgagcccgtctggccccgcgcacgtggtgccgtttgctgcaggtgtccggcggaacctggggcatgcacctgcgcaacaccatacgggaggtggaggctcgattcgacgccaccgcggaacccgtgtgcaagatccttgtttggagaccagacggtacggcccggagtgtgatcttagtaatctcgagattcatctcagcgcgacaagcgatgatgaaatctccgatgccaccgatctggaggccgccggttcggaccacacgctcgcgtcccagtccgacacggaggatgccccctcccccgttacgctggaaaccccagaaccccgcgggtccctcgctgtgcgtctggaggatgagtttggggagtttgactggaccccccaggagggctcccagccctggctgtctgcggtcgtggccgataccagctccgtggaacgcccgggcccatccgattctggggcgggtcgcgccgcagaagaccgcaagtgtctggacggctgccggaaaatgcgcttctccaccgcctgcccctatccgtgcagcgacacgtttctccggccgtgagtccggtcgccccgaccccatgtatgtccccaaaataaaagaccaaaatcaaagcgtttgtcccagcgtataatggcgggaagggcggagagaaacagaccacgcggacatggggggtgtttgggggtttattggcaccgggggctaaagggtggtaaccggatagcagatgtgaggaagtcggggccgttcgccgcgaacggcgatcagagggtcagtttcttgcggaccacggcccggcgatgtgggttgctcgtctgggacctcgggcatgcccatacacgcacaacacggacgccgcaccggatgggacgtcgtaagggggcctggggtagctgggtggggtttgtgcagagcaatcagggaccgcagccagcgcatacaatcgcgctcccgtccgtttgtcccgggcagtaccacgccgtactggtattcgtaccggctgagcagggtctccagggggtggttgggggccgcggggaacggggtccacgccacggtccactcgggcaaaaaccgagtcggcacggcccacggttctcccacccacgcgtctggggtcttgatggcgataaatcttaccccgagccggattttttgggcgtattcgagaaacggcacacacagatccgccgcgcctaccacccacaagtggtagaggcgaggggggctgggttggtctcggtgcagcagtcggaagcacgccacggcgtccacgacctcggtgctctccaaggggctgtcctccgcaaacaggcccgtggtggtgtttggggggcagcgacaggacctagtgcgcacgatcgggcgggtgggtttgggtaagtccatcagcggctcggccaaccgtcgaaggttggccggacgaacgacgaccggggtacccaggggttctgatgccaaaatgcggcactgcctaagcaggaagctccacagggccgggcttgcgtcgacggaagtccggggcagggcgttgttctggtcaaggagggtcattacgttgacgacaacaacgcccatgttggtatattacaggcccgtgtccgatttggggcacttgcagatttgtaaggccacgcacggcggggagacaggccgacgcgggggctgctctaaaaatttaagggccctacggtccacagacccgccttcccgggggggcccttggagcgcccggcagcggaggcgtccgggggaggggagggtgatttacgggggggtaggtcagggggtgggtcgtcaaactgccgctccttaaaaccccggggcccgtcgttcggggtgctcgaggaggcactcacggtgcggcgcatggcctgtcgtaagattgtcgcgatacgggggacagggcaggaggcaggaggaggccgtcccgccggagacaaagccgtcccgggtgtttcctcatggccccttttataccccagccgaggacgcgtgcctggactccccgcccccggagacccccaaaccttcccacaccacaccacccagcgaggccgagcgcctgtgtcatctgcaggagatccttgcccagatgtacggaaaccaggactaccccatagaggacgaccccagcgcggatgccgcggacgatgtcgacgaggacgccccggacgacgtggcctatccggaggaatacgcagaggagctttttctgcccggggacgcgaccggtccccttatcggggccaacgaccacatccctcccccgtgtggcgcatctccccccggtatacgacgacgcagccgggatgagattggggccacgggatttaccgcggaagagctggacgccatggacagggaggcggctcgagccatcagccgcggcggcaagcccccctcgaccatggccaagctggtgactggcatgggctttacgatccacggagcgctcaccccaggatcggaggggtgtgtctttgacagcagccatccagattacccccaacgggtaatcgtgaaggcggggtggtacacgagcacgagccacgaggcgcgactgctgaggcgactggaccacccggcgatcctgcccctcctggacctgcatgtcgtctccggggtcacgtgtctggtcctccccaagtaccaggccgacctgtatacctatctgagtaggcgcctgaacccactgggacgcccgcagatcgcagcggtctcccggcagctcctaagcgccgttgactacattcaccgccagggcattatccaccgcgacattaagaccgaaaatatttttattaacacccccgaggacatttgcctgggggactttggcgccgcgtgcttcgtgcagggacccgatccagccccttcccctacggaatcgccggaaccatcgacacccacgcccccgaggtcctggccggggatcc.

The α4 promoter gene with the unique short sequence containing the ICP47ORF is found at nucleotides 144876-146834, and has the followingsequence:

(SEQ ID NO: 8) ggatccgtgtcggcagccgcgctccgtgtggacgatcggggcgtcctcgggctcatatagtcccaggggccggcgggaaggaggagcagcggaggccgccggccccccgcccccccggcgggcccaccccgaacggaattccattatgcacgaccccgccccgacgccggcacgccgggggcccgtggccgcggcccgttggtcgaacccccggccccgcccatccgcgccatctgccatgggcggggcgcgagggcgggtgggtccgcgccccgccccgcatggcatctcattaccgcccgatccggcggtaccgcttccgaccgcatgctaacgaggaacgggcagggggcggggcccgggccccgacttcccggttcggcggtaatgagatacgagccccgcgcgcccgttggccgtccccgggccccccggtcccgcccgccggacgccgggaccaacgggacggcgggcggcccaagggccgcccgccttgccgcccccccattggccggcgggcgggaccgccccaagggggcggggccgccgggtaaaagaagtgagaacgcgaagcgttcgcacttcgtcccaatatatatatattattagggcgaagtgcgagcactggcgccgtgcccgactccgcgccggccccgggggcgggcccgggcggcggggggcgggtctctccggcgcacataaaggcccggcgcgaccgacgcccgcagacggcgccggccacgaacgacgggagcggctgcggagcacgcggaccgggagcgggagtcgcagagggccgtcggagcggacggcgtcggcatcgcgacgccccggctcgggatcgggatcgcatcggaaagggacacgcggacgcgggggggaaagacccgcccaccccacccacgaaacacaggggacgcaccccgggggcctccgacgacagaaacccaccggtccgccttattgcacgggtaagcaccttgggtgggcggaggagggggggacgcgggggcggaggaggggggacgcgggggcggaggaggggggacgcgggggcggaggaggggggacgcgggggcggaggaggggggacgcgggggcggaggagggggctcacccgcgttcgtgccttcccgcaggaggaacgtcctcgtcgaggcgaccggcggcgaccgttgcgtggaccgcttcctgctcgtcgggcggggggaagccactgtggtcctccgggacgttttctggatggccgacataccccaggcgcttttgcgccttgtgtaaaagcgcggcgtcccgctctccgatccccgcccctgggcacgcgcaagcgcaagcgcccttcccgccccctctcatcggagtctgaggtagaatccgatacagccttggagtctgaggtcgaatccgagacagcatcggattcgaccgagtctggggaccaggatgaagccccccgcatcggtggccgtagggccccccggaggcttggggggcggttttttctggacatgtcggcggaatccaccacggggacggaaacggatgcgtcggtgtcggacgaccccgacgacacgtccgactggtcttatgacgacattcccccacgacccaagcgggcccgggtaaacctgcggctcacgagctctcccgatcggcgggatggggttatttttcctaagatggggcgggtccggtctacccgggaaacgcagccccgggcccccaccccgtcggccccaagcccaaatgcaatgctacggcgctcggtgcgccaggcccagaggcggagcagcgcacgatggacccccgacctgggctacatgcgccagtgtatcaatcagctgtttcgggtcctgcgggtcgcccgggacccccacggcagtgccaaccgcctgcgccacctgatacgcgactgttacctgatgggatactgccgagcccgtctggccccgcgcacgtggtgccgtttgctgcaggtgtccggcggaacctggggcatgcacctgcgcaacaccatacgggaggtggaggctcgattcgacgccaccgcggaacccgtgtgcaagatccttgtttggagaccagacggtacggcccggagtgtgatcttagtaatctcgagattcatctcagcgcgacaagcgatgatgaaatctccgatgccaccgatctggaggccgccggttcggaccacacgctcgcgtcccagtccgacacggaggatgccccctcccccgttacgctggaaaccccagaaccccgcgggtccctcgctgtgcgtctggaggatgagtttggggagtttgactggaccccccaggagggctcccagccctggctgtctgcggtcgtggccgataccagctccgtggaacgcccgggcccatccgattctggggcgggtcgcgccgcagaagaccgcaagtgtctggacggctgccggaaaatgcgcttctccaccgcctgcccctatccgtgcagcgacacgtttctccggccgtgagtccggtcgccccgacccccttgtatgtccccaaaataaaagaccaaaatcaaagcgtttgtcccagcgtcttaatggcgggaagggcggagagaaacagaccacgcggacatggggggtgtttgggggtttattggcaccgggggctaaagggtggtaaccggatagcagatgtgaggaagtcggggccgttcgccgcgaacggcgatcagagggtcagatcttgcggaccacggcccggcgatgtgggagctcgtctgggacctcgggcatgcccatacacgcacaacacggacgccgcaccggatgggacgtcgtaagggggcctggggtagctgggtggggtttgtgcagagcaatcagggaccgcagccagcgcatacaatcgcgctcccgtccgtttgtcccgggcagtaccacgccgtactggtattcgtaccggctgagcagggtctccagggggtggttgggggccgcggggaacggggtccacgccacggtccactcgggcaaaaaccgagtcggcacggcccacggttctcccacccacgcgtctggggtcttgatggcgataaatcttaccccgagccggattttttgggcgtattcgagaaacggcacacacagatccgccgcgcctaccacccacaagtggtagaggcgaggggggctgggttggtctcggtgcagcagtcggaagcacgccacggcgtccacgacctcggtgctctccaaggggctgtcctccgcaaacaggcccgtggtggtgatggggggcagcgacaggacctagtgcgcacgatcgggcgggtgggtttgggtaagtccatcagcggctcggccaaccgtcgaaggttggccggacgaacgacgaccggggtacccaggggttctgatgccaaaatuggcactgcctaagcaggaagctccacagggccgggcttgcgtcgacggaagtccggggcagggcgttgttctggtcaaggagggtcattacgttgacgacaacaacgcccatgttggtatattacaggcccgtgtccgatttggggcacttgcagatttgtaaggccacgcacggcggggagacaggccgacgcgggggctgctctaaaaatttaagggccctacggtccacagacccgccttcccgggggggcccttggagcgaccggcagcggaggcgtccgggggaggggagggtgatttacgggggggtaggtcagggggtgggtcgtcaaactgccgctccttaaaaccccggggcccgtcgttcggggtgctcgttggttggcactcacggtgcggcgaatggcctgtcgtaagttagtcgcgtttacgggggacagggcaggaggaaggaggaggccgtcccgccggagacaaagccgtcccgggtgtacctcatggccccttttataccccagccgaggacgcgtgcctggactccccgcccccggagacccccaaaccttcccacaccacaccacccagcgaggccgagcgcctgtgtcatctgcaggagatccttgcccagatgtacggaaaccaggactaccccatagaggacgaccccagcgcggatgccgcggacgatgtcgacgaggacgccccggacgacgtggcctatccggaggaatacgcagaggagctattctgcccggggacgcgaccggtccccttatcggggccaacgaccacatccctcccccgtgtggcgcatctccccccggtatacgacgacgcagccgggatgagattggggccacgggatttaccgcggaagagctggacgccatggacagggaggcggctcgagccatcagccgcggcggcaagcccccctcgaccatggccaagctggtgactggcatgggctttacgatccacggagcgctcaccccaggatcggaggggtgtgtctttgacagcagccatccagattacccccaacgggtaatcgtgaaggcggggtggtacacgagcacgagccacgaggcgcgactgctgaggcgactggaccacccggcgatcctgcccctcctggacctgcatgtcgtctccggggtcacgtgtctggtcctccccaagtaccaggccgacctgtatacctatctgagtaggcgcctgaacccactgggacgcccgcagatcgcagcggtctcccggcagctcctaagcgccgttgactacattcaccgccagggcattatccaccgcgacattaagaccgaaaatatttttattaacacccccgaggacatttgcctgggggactttggcgccgcgtgcttcgtgcagggttcccgatcaagccccttcccctacggaatcgccggaaccatcgacaccaacgcccccgaggtcctggccggggatcc.

The α22 promoter gene is found at nucleotides 131249-132604, and has thefollowing

(SEQ ID NO: 9) gtcgacgcggaactagcgcggaccggtcgatgcttgggtgggaaaaaggacagggacggccgatccccctcccgcgcttcgtccgcgtatcggcgtcccggcgcggcgagcgtctgacggtctgtctctggcggtcccgcgtcgggtcgtggatccgtgtcggcagccgcgctccgtgtggacgatcggggcgtcctcgggctcatatagtcccaggggccggcgggaaggaggagcagcggaggccgccggccccccgcccccccggcgggcccaccccgaacggaattccattatgcacgaccccgccccgacgccggcacgccgggggcccgtggccgcggcccgttggtcgaacccccggccccgcccatccgcgccatctgccatgggcggggcgcgagggcgggtgggtccgcgccccgccccgcatggcatctcattaccgcccgatccggcggtttccgcttccgttccgcatgctaacgaggaacgggcagggggcggggcccgggccccgacttcccggttcggcggtaatgagatacgagccccgcgcgcccgttggccgtccccgggccccccggtcccgcccgccggacgccgggaccaacgggacggcgggcggcccaagggccgcccgccttgccgcccccccattggccggcgggcgggaccgccccaagggggcggggccgccgggtaaaagaagtgagaacgcgaagcgttcgcacttcgtcccaatatatatatattattagggcgaagtgcgagcactggcgccgtgcccgactccgcgccggccccgggggcgggcccgggcggcggggggcgggtctctccggcgcacataaaggcccggcgcgaccgacgcccgcagacggcgccggccacgaacgacgggagcggctgcggagcacgcggaccgggagcgggagtcgcagagggccgtcggagcggacggcgtcggcatcgcgacgccccggctcgggatcgggatcgcatcggaaagggacacgcggacgcgggggggaaagacccgcccaccccacccacgaaacacaggggacgcaccccgggggcctccgacgacagaaacccaccggtccgccttttttgcacgggtaagcaccttgggtgggcggaggagggggggacgcgggggcggaggaggggggacgcgggggcggaggaggggggacgcgggggcggaggaggggggacgcgggggcggaggaggggggacgcgggggcggaggagggggctcacccgcgttcgtgccttcccgcaggaggaacgtcctcgtcgaggcgaccggcggcgaccgttgcgtggaccgcttcctgctc gtcggg.

The α27 promoter gene is found at nucleotides 111990-113647, and has thefollowing sequence:

(SEQ ID NO: 10) cctgcagcaaagcctgtcgtgtctgcgctttaagcacggccgggcgagtcgcgccacggcgcggacattcgtcgcgctgagcgtcggggccaacaaccgcctgtgcgtgtccttgtgtcagcagtgctttgccgccaaatgcgacagcaaccgcctgcacacgctgtttaccattgacgccggcacgccatgctcgccgtccgttccctgcagcacctctcaaccgtcgtcttgataacggcgtacggcctcgtgctcgtgtggtacaccgtcttcggtgccagtccgctgcaccgatgtatttacgcggtacgccccaccggcaccaacaacgacaccgccctcgtgtggatgaaaatgaaccagaccctattgatctgggggccccgacgcacccccccaacgggggctggcgcaaccacgcccatatctgctacgccaatcttatcgcgggtagggtcgtgcccttccaggtcccacctgacgccatgaatcgtcggatcatgaacgtccacgaggcagttaactgtctggagaccctatggtacacacgggtgcgtctggtggtcgtagggtggacctgtatctggcgttcgtcgccctccaccaacgccgatgtatgtttggcgtcgtgagtcccgcccacaagatggtggccccggccacctacctcttgaactacgcaggccgcatcgtatcgagcgtgttcctgcagtacccctacacgaaaattacccgcctgctctgcgagctgtcggtccagcggcaaaacctggttcagttgtttgagacggacccggtcaccttcttgtaccaccgccccgccatcggggtcatcgtaggctgcgagttgatgctacgctttgtggccgtgggtctcatcgtcggcaccgctttcatatcccggggggcatgtgcgatcacataccccctgtactgaccatcaccacctggtgttttgtctccaccatcggcctgacagagctgtattgtattctgcggcggggcccggcccccaagaacgcagacaaggccgccgccccggggcgatccaaggggctgtcgggcgtctgcgggcgctgctgttccatcatcctctcgggcatcgcagtgcgattgtgttatatcgccgtggtggccggggtggtgctcgtggcgcttcactacgagcaggagatccagaggcgcctgtttgatgtatgacgtcacatccaggccggcggaaaccgtaacggcatatgcaaattggaaactgtcctgtcttggggcccacccacccgacgcgtcatatgcaaatgaaaatcggtcccccgaggccacgtgtagcctggatcccaacgaccccgcccatgggtcccaattggccgtcccgttaccaagaccaacccagccagcgtatccacccccgcccgggtccccgcggaagcggaacggggtatgtgatatgctaattaaatacatgccacgtacttatggtgtctgattggtccttgtctgtgccggaggtggggcgggggccccgcccggggggcggaacgaggaggggtttgggagagccggccccggcaccacgggtataaggacatccaccacccggccggtggtggtgtgcagccgtgttccaaccacggtcacgcttcggtgcctctc cccga.

The α47 promoter gene is found at nucleotides 145585-146984, and has thefollowing sequence:

(SEQ ID NO: 11) cccgacgagcaggaagcggtccacgcaacggtcgccgccggtcgcctcgacgaggacgacctcctttgggaaggcacgaacgcgggtgagcccctcctccgcccccgcgtcccccctcctccgcccccgcgtcccccctcctccgcccccgcgtcccccctcctccgcccccgcgtccccctcctccgcccccgcgtcccccctcctccgcccccgcgtcccccctcctccacccccgcgtccccctcctccgcccacccaaggtgcttacccgtgcaaaaaaggcggaccggtgggatctgtcgtcggaggcccccggggtgcgtcccctgtgatcgtgggtggggtgggcgggtctttcccccccgcgtccgcgtgtccctttccgatgcgatcccgatcccgagccggggcgtcgcgatgccgacgccgtccgctccgacggccctctgcgactcccgctcccggtccgcgtgctccgcagccgctcccgtcgttcgtggccggcgccgtctgcgggcgtcggtcgcgccgggcctttatgtgcgccggagagacccgccccccgccgcccgggcccgcccccggggccggcgcggagtcgggcacggcgccagtgctcgcacttcgccctaataatatatatatattgggacgaagtgcgaacgcttcgcgttctcacttcttttacccggcggccccgcccccttggggcggtcccgcccgccggccaatgggggggcggcaaggcgggcggcccttgggccgcccgccgtcccgttggtcccggcgtccggcgggcgggaccggggggcccggggacggccaacgggcgcgcggggctcgtatctcattaccgccgaaccgggaagtcggggcccgggccccgccccctgcccgttcctcgttagcatgcggaacggaagcggaaaccgccggatcgggcggtaatgagatgccatgcggggcggggcgcggacccacccgccctcgcgccccgcccatggcagatggcgcggatgggcggggccgggggttcgaccaacgggccgcggccacgggcccccggcgtgccggcgtcggggcggggtcgtgcataatggaattccgttcggggtgggcccgccgggggggcggggggccggcggcctccgctgctcctccttcccgccggcccctgggactatatgagcccgaggacgccccgatcgtccacacggagcgcggctgccgacacggatccacgacccgacgcgggaccgccagagacagaccgtcagacgctcgccgcgccgggacgccgatacgcggacgaagcgcgggagggggatcggccgtccctgtcctttttcccacccaagcatcgaccggtccgcgctagttccgcgtcgac.

Mutations may be made in the variant HSV by homologous recombinationmethods well known to those skilled in the art. For example, HSV genomicDNA is transfected together with a vector, preferably a plasmid vector,comprising the mutated sequence flanked by homologous HSV sequences. Themutated sequence may comprise a deletion(s), insertion(s) orsubstitution(s), all of which may be constructed by routine techniques.Insertions may include selectable marker genes, for example lacZ orgreen fluorescent protein (GFP), which may be used for screeningrecombinant viruses, for example, β-galactosidase activity orfluorescence. In a preferred embodiment, the selectable marker is theIE-Us11 gene.

The generation of variant HSV having intact endogenous Us11 and Us12genes and a Us11 gene operatively associated with an IE promoterinserted into the γ₁34.5 locus is described in detail in U.S. Pat. No.7,731,952 by Mohr et al. The instant invention provides improved variantHSV that are based on the variant HSV described in U.S. Pat. No.7,731,952, and that further comprise one or more genes encodingheterologous polypeptides. As described herein, preferably, theheterologous peptides are inserted into the same region of the HSVgenome as the IE-Us11 gene (i.e., the γ₁34.5 locus).

The heterologous polypeptide encoding genes described herein may beinserted into the viral genome by any suitable technique such ashomologous recombination of HSV strains with, for example, plasmidvectors carrying the gene flanked by HSV sequences. For example, a geneencoding a heterologous polypeptide can be inserted into a geneexpression cassette, according to the methods described in U.S. Pat. No.7,731,952 for insertion of IE-Us11. As described in Examples 2 and 3,below, for example, the heterologous polypeptide encoding gene may befused to a CMV or EF1α promoter in an expression cassette, and thisexpression cassette may be inserted into a targeting vector in place ofγ₁34.5. The expression cassette may be designed to have flankingsequences that mediate homologous recombination into the γ₁34.5 locus.Preferably, the one or more genes encoding a heterologous polypeptideare inserted at the same site in the HSV genome as the TE-Us11 gene, forexample, by including them on the same polynucleotide cassette as theIE-Us11 gene. However, the one or more genes encoding a heterologouspolypeptide may also be inserted at other sites. In variant HSVcomprising two genes encoding heterologous polypeptides, expression ofeach gene may be driven by separate promoters, for example a CMVpromoter and an EF1α promoter, or two CMV promoters or two EF1αpromoters arranged in opposite orientation or from a single promoter,e.g., one CMV or EF1α promoter driving expression of both genes. Whereboth heterologous polypeptide encoding genes are expressed from a singlepromoter, the genes may be separated by an internal ribosome entry site(IRES). The genes may also be expressed as a translational fusion suchthat the fused polypeptide retains both activities of the separate genes(e.g., prodrug activation and cell to cell fusion, prodrug activationand immunomodulatory activity or cell to cell fusion andimmunomodulatory activity) such that the fused polypeptides are cleavedfollowing expression by a protease either in cis or in trans to thefused polypeptide. It is also possible that the fused polypeptides arenot separated by a cleavage site but still retain the activities of theseparate genes.

The transcribed sequences of the inserted genes are preferably operablyassociated with control sequences permitting expression of the genes ina tumor cell. A control sequence typically comprises a promoter allowingexpression of the gene operably associated therewith and signal fortermination of transcription. The promoter is selected from promoterswhich are functional in mammalian, preferably human tumor cells. Thepromoter may be derived from promoter sequences of a eukaryotic gene.For example, the promoter may be derived from the genome of a cell inwhich expression of the heterologous gene is to occur, preferably amammalian tumor cell, more preferably a human tumor cell. With respectto eukaryotic promoters, they may be promoters that function in aubiquitous manner (such as promoters of β-actin, tubulin) or,alternatively, in a tumor-specific manner. They may also be promotersthat respond to specific stimuli, for example promoters that bindsteroid hormone receptors. Viral promoters may also be used, for examplethe Moloney murine leukaemia virus long terminal repeat (MMLV LTR)promoter or other retroviral promoters such as that derived from Roussarcoma virus (RSV), the human or mouse cytomegalovirus (CMV) IEpromoter or promoters of herpes virus genes including those drivingexpression of the latency associated transcripts.

Expression cassettes and other suitable constructs comprising theprodrug converting Enzyme encoding gene, gene encoding a polypeptidecapable of promoting cell to cell fusion and/or immunomodulatory geneand control sequences can be made using routine cloning techniques knownto persons skilled in the art (see, for example, Sambrook et al., 1989,Molecular Cloning—A laboratory manual; Cold Spring Harbor Press).

It may also be advantageous for the promoter(s) to be inducible so thatthe levels of expression of the genes can be regulated during thelife-time of the tumor cell. Inducible means that the levels ofexpression obtained using the promoter can be regulated. For example, avirus of the invention may further comprise a heterologous gene encodingthe tet repressor/VP16 transcriptional activator fusion protein underthe control of a strong promoter (e.g., the CMV IE promoter) and theprodrug converting, cell to cell fusion or immunomodulatory or othergene may be under the control of a promoter responsive to the tetrepressor VP16 transcriptional activator fusion protein previouslyreported (see, Gossen et al., “Tight control of gene expression inmammalian cells by tetracycline-responsive promoters,” Proc. Natl. Acad.Sci. USA, 89: 5547-5551, 1992, Gossen et al., “Transcriptionalactivation by tetracyclines in mammalian cells,” Science, 268:1766-1769, 1995). Thus, in this example, expression of the gene(s) woulddepend on the presence or absence of tetracycline.

In a specific embodiment, the heterologous polypeptide encoding gene,e.g., in a gene expression cassette, may be inserted, preferably intothe γ₁34.5 locus, such that it has the same orientation as the IE-Us11gene or the opposite orientation. Furthermore, the heterologouspolypeptide may be inserted either upstream or downstream of the IE-Us11gene. When two heterologous polypeptide encoding genes are present, eachgene may be present in either orientation, i.e., the first and secondheterologous polypeptide encoding genes can each have either the same oropposite orientation as the IE-Us11 gene. Preferably, in a variant HSVcomprising two heterologous polypeptide encoding genes, one of thosegenes is expressed from a CMV promoter, and the other from an EF1αpromoter, wherein the promoters (and genes) are placed in a back-to-backorientation with respect to each other and inserted into the HSV genomeso as to replace the genes encoding ICP34.5. However, other promotersmay also be used. The polynucleotide cassettes used to make the variantHSV of the invention are shown in FIGS. 2, 3A-3D, and 4. Theheterologous polypeptide genes may also be inserted into the viralgenome at other location(s) in the viral genome, however, provided thatthe desired oncolytic properties and, preferably, immune evasionabilities of the variant HSV, are retained.

Variant HSV of the invention can also encode multiple heterologous genes(e.g., prodrug converting enzyme encoding genes, genes encoding apolypeptide capable of promoting cell to cell fusion and/orimmunomodulatory genes). Variant HSV of the invention may comprise oneor more additional genes, for example from 1, 2 to 3, 4 or 5 additionalgenes. The additional gene(s) may be further copies of the heterologouspolypeptide encoding gene(s). The additional gene(s) may encode one ormore different prodrug converting gene, one or more different fusiogenicgene and/or one or more different immunomodulatory gene and/or one ormore matrix modifying enzymes. The additional gene(s) may encode othergene(s) intended to enhance the therapeutic effect.

More than one gene and associated control sequences could be introducedinto a particular HSV either at a single site or at multiple sites inthe virus genome. Alternatively pairs of promoters (the same ordifferent promoters) facing in opposite orientations away from eachother, each driving the expression of a gene may be used.

In certain embodiments, a gene encoding a heterologous polypeptide is aprodrug activating enzyme, a heterologous gene encoding a polypeptidecapable of causing cell to cell fusion or a heterologous gene encodingan immunomodulatory polypeptide. In a preferred embodiment, the variantHSV comprises at least two (2) genes encoding heterologous polypeptides.

A prodrug activating polypeptide can be a cytosine deaminase enzyme,which is capable of converting the inactive prodrug 5-fluorocytosine tothe active drug 5-flurouracil. Various cytosine deaminase genes areavailable including those of bacterial origin and of yeast origin. Asecond gene, typically a gene encoding a second enzyme, may be used toenhance the prodrug conversion activity of the cytosine deaminase gene.For example, the second gene may encode a uracilphosphoribosyltransferase.

Any suitable fusogenic gene encoding a polypeptide capable of causingcell to cell fusion may be used. Preferably the polypeptide capable ofcausing cell to cell fusion is selected from a modified retroviralenvelope glycoprotein, such as an envelope glycoprotein derived fromgibbon ape leukaemia virus (GALV) or human endogenous retrovirus W, afusogenic F or H protein from measles virus and the vesicular stomatitisvirus G protein. More preferably, the polypeptide capable of causingcell to cell fusion is a GALV fusogenic glycoprotein (see, Simpson etal. (2006) “Combination of a Fusogenic Glycoprotein, Prodrug Activation,and Oncolytic Herpes Simplex Virus for Enhanced Local Tumor Control.”Cancer Res; 66:9: 4835-4842).

The immunomodulatory gene may be any gene encoding a polypeptide that iscapable of modulating an immune response. The polypeptide capable ofmodulating an immune response may be a polypeptide capable of inhibitingantigen presentation on class I MHC molecules, for example, a TAPinhibitor (such as certain UL49.5 polypeptides (e.g., from BHV), humanCMV US3 and US6, HSV Us12/ICP47, EBV, or BNLF2a) or a class I MHCmolecule maturation inhibitor (e.g., murine CMV mK3, human CMV US2 andUS11 (not related to HSV Us11), and varicella zoster virus ORF66). Thepolypeptide capable of modulating an immune response also may be acytokine such as, but not limited to, GM-CSF, TNF-α, an interleukin (forexample IL12), an interferon (such as IFNγ) a chemokine such as RANTESor a macrophage inflammatory protein (MIP) (for example, MIP-3), oranother immunomodulatory molecule such as B7.1 (CD80), B7.2 (CD86) orCD40L.

The polypeptide capable of causing cell to cell fusion may also becapable of modulating an immune response. For example, GALV is capableof modulating an immune response. Variant HSV of the invention may thusbe used to deliver the genes to a cell in vivo where they will beexpressed.

Non-limiting examples of TAP-inhibitor genes include UL49.5, e.g., frombovine herpesvirus (BHV), which is capable of inhibiting mouse and humanTAP (van Hall et al., J. Immunology (2007) 178:657-662). UL49.5polypeptides can also be derived from pseudorabies virus (PRV) andequine herpesvirus 1 and 4 (EHV-1 and EHV-4). These UL49.5 proteinsinterfere with MHC class I antigen presentation by blocking the supplyof antigenic peptides through inhibition of TAP and are active on rodentTAP, such as murine TAP. Other examples of TAP inhibitors include UL49.5polypeptides from bubaline herpesvirus 1, cervid herpesvirus 1, felidherpesvirus 1, (see, Verweij et al. 2011 “Structural and functionalanalysis of the TAP-inhibiting UL49.5 proteins of varicelloviruses.” MolImmunol. July 15 Epub) and BNLF2a and ICP47. It is noted that UL49.5homolog from HSV-1 and HSV-2 do not inhibit TAP [see, Koppers-Lalic, D.et al. (2008) PLoS; 4(5): e1000080].

Non-limiting examples of marix modifying enzymes are: matrixmetalloproteinases such as collagenases, gelatinases and stromelysins,relaxin, bacterial collagenase and chondroitinase ABC 1.

Although the invention is not limited by any particular theory ormechanism of action, the insertion of the gene for mammalian GM-CSF intothe genome of the variant HSV of the invention can enhance anti-tumorresponses both locally and at sites distant to where the variant HSV isinjected by stimulating T-cell mediated immune responses. GM-CSF is theprincipal mediator of proliferation, maturation, and migration ofdendritic cells, the most potent antigen presenting cells of the immunesystem. Dendritic cells display antigens on their surface in conjunctionwith class II major histocompatibility complex (MHC-II). Once presentedon MHC class II molecules, the antigen can be recognized by helper CD4+T cells, which provide support for the development of B cells andcytolytic CD8+ T cells. Expression of GM-CSF in the local tumorenvironment serves to achieve several biologic goals: (a) induces localinflammation, (b) enhances dendritic cell activity, and (c) increasesHLA class II expression. Further, in certain embodiments, cytokineshaving similar activity as GM-CSF, as described above, are alsocontemplated for use in the present invention. Under such increasedimmune recruitment and activation conditions, immunomodulatorypolypeptides that lead to enhanced immune recruitment and activationcould be deleterious to viral infection and spread. For this reason, theenhanced immune evasion capabilities of the variant HSV of the invention(provided by the heterologous gene encoding an inhibitor of class I MHCmolecule antigen presentation (e.g., TAP inhibitor)) are particularlyimportant for promoting viral replication, spread and efficacy.

In other embodiments, using standard molecular and virologicaltechniques, an oncolytic virus strain (e.g., OV-2711) may be modified tocreate novel, cancer-specific variant HSV of the present invention. Forexample, variant HSV may be engineered according to the invention wherePI-3-kinase signaling is constitutively activated, e.g., by deleting thevirus-encoded Akt mimic Us3. Alternatively, a key viral surfaceglycoprotein may be altered, such that the virus preferentially enterscells within the urothelium. Variant HSV of the invention may haveeither one or both of these modifications, and their oncolytic activitymay be evaluated in both cell culture and animal models well known inthe art.

Pharmaceutical Compositions

Pharmaceutical compositions include an active agent and apharmaceutically acceptable carrier, excipient, or diluent.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water or aqueoussolution saline solutions and aqueous dextrose and glycerol solutionsare preferably employed as carriers, particularly for injectablesolutions. Alternatively, the carrier can be a solid dosage formcarrier, including but not limited to one or more of a binder (forcompressed pills), a glidant, an encapsulating agent, a flavorant, and acolorant. Suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

When formulated in a pharmaceutical composition, a therapeutic compoundof the present invention can be admixed with a pharmaceuticallyacceptable carrier or excipient. As used herein, the phrase“pharmaceutically acceptable” refers to molecular entities andcompositions that are generally believed to be physiologically tolerableand do not typically produce an allergic or similar untoward reaction,such as gastric upset, dizziness and the like, when administered to ahuman.

The term “pharmaceutically acceptable derivative” as used herein meansany pharmaceutically acceptable salt, solvate or prodrug, e.g. ester, ofa compound of the invention, which upon administration to the recipientis capable of providing (directly or indirectly) a compound of theinvention, or an active metabolite or residue thereof. Such derivativesare recognizable to those skilled in the art, without undueexperimentation. Nevertheless, reference is made to the teaching ofBurger's Medicinal Chemistry and Drug Discovery, 5th Edition, Vol 1:Principles and Practice, which is incorporated herein by reference tothe extent of teaching such derivatives. Preferred pharmaceuticallyacceptable derivatives are salts, solvates, esters, carbamates, andphosphate esters. Particularly preferred pharmaceutically acceptablederivatives are salts, solvates, and esters. Most preferredpharmaceutically acceptable derivatives are salts and esters.

While it is possible to use a composition provided by the presentinvention for therapy as is, it may be preferable to administer it in apharmaceutical formulation, e.g., in admixture with a suitablepharmaceutical excipient, diluent, or carrier selected with regard tothe intended route of administration and standard pharmaceuticalpractice. Accordingly, in one aspect, the present invention provides apharmaceutical composition or formulation comprising at least one activecomposition, or a pharmaceutically acceptable derivative thereof, inassociation with a pharmaceutically acceptable excipient, diluent,and/or carrier. The excipient, diluent and/or carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

The compositions of the invention can be formulated for administrationin any convenient way for use in human or veterinary medicine.

For human therapy, the pharmaceutical compositions, including each ofthe active agents, will be prepared in accordance with goodmanufacturing process (GMP) standards, as set by the Food & DrugAdministration (FDA). Quality assurance (QA) and quality control (QC)standards will include testing for purity and function and otherstandard measures.

A preferred delivery vehicle is any chemical entity that ensuresdelivery of a variant HSV to a tumor cell in a selective manner,achieves sufficient concentration of variant HSV in the tumor cell. Thiscan include, without limitation, standard pharmaceutical dosage formsfor the delivery of a virus (e.g., solutions, suspensions, emulsions)with or without controlled release. Other dosage forms, e.g., soliddosage forms such as, but not limited to, crystals or beads may also beused.

Therapeutic Uses

In certain embodiments, the present invention provides methods forkilling tumor cells in a subject and for treating cancers, including, inpreferred embodiments, bladder cancer. In one embodiment, an oncolyticor other virus of the invention can be used in a “stand alone” ormonotherapy to treat such cancers. However, the invention also includesmethods and compositions where an oncolytic or other virus of theinvention is combined with at least one other therapeutic substance ortreatment modality for treating cancer. In a preferred embodiment, theother therapeutic substance is cisplatin. However, any chemical or otheragent used to treat bladder or other cancers can be used. Non-limitingexamples of cancers that can be treated using the variant HSV of theinvention include, e.g., prostate cancer, glioma, melanoma, coloncancer, ovarian cancer, breast cancer, head/neck cancer, and includingall solid tumors.

The specific conditions (e.g., appropriate pharmaceutical carrier,dosage, site and route of administration, etc.) under which a variantHSV-containing composition of the invention should be administered inorder to be effective for killing tumor cells or for treating cancer isan individual can be determined, e.g., by the individual's physician.

Individuals that can be treated according to the methods describedherein include mammals, such as rodents, dogs, cats, etc., and includinghumans.

Variant HSV of the invention may be used in a method of treating thehuman or animal body. In particular, viruses of the invention may beused in methods of cancer therapy. Preferably, variant HSV of theinvention are used in the oncolytic treatment of cancer. Viruses of theinvention may be used in the therapeutic treatment of any solid tumor ina mammal, preferably a human. For example viruses of the invention maybe administered to a subject with prostate, breast, lung, liver, renalcell, endometrial, bladder, colon or cervical carcinoma; adenocarcinoma;melanoma; lymphoma; glioma; sarcomas such as soft tissue and bonesarcomas; or cancer of the head and neck, and, preferably, bladdercancer.

The term “cancer” refers to all types of cancer, neoplasm or malignanttumors found in mammals, including leukemia, carcinomas and sarcomas.Exemplary cancers include cancer of the breast, brain, cervix, colon,head & neck, liver, kidney, lung, non-small cell lung, melanoma,mesothelioma, ovary, sarcoma, stomach, uterus and Medulloblastoma.Additional examples include, Hodgkin's Disease, Non-Hodgkin's Lymphoma,multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma,primary thrombocytosis, primary macroglobulinemia, primary brain tumors,malignant pancreatic insulinoma, malignant carcinoid, urinary bladdercancer, premalignant skin lesions, testicular cancer, lymphomas, thyroidcancer, neuroblastoma, esophageal cancer, genitourinary tract cancer,malignant hypercalcemia, endometrial cancer, adrenal cortical cancer,neoplasms of the endocrine and exocrine pancreas, and prostate cancer.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas include, for example, acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoidcarcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,carcinoma ex ulcere, carcinoma fibrosum, gelatiniformi carcinoma,gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypemephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

In certain embodiments, the compositions provided herein are useful forkilling tumor cells selected from the group consisting of astrocytoma,oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma,Schwannoma, neurofibrosarcoma, medulloblastoma, melanoma cells,pancreatic cancer cells, prostate carcinoma cells, breast cancer cells,lung cancer cells, colon cancer cells, hepatoma cells, mesothelioma andepidermoid carcinoma cells.

In one embodiment, the cancer to be treated is bladder cancer. Bladdercancer (BC) is the fifth most common human malignancy and the secondmost common genitourinary tumor. Intensive surveillance withcystoscopies, urinary cytologies, and frequent tumor resections underanesthesia make BC the most costly malignancy to treat. Despite advancesin intravesical and systemic chemotherapy, immunotherapy, and surgery,the efficacy of present treatment options remains limited and theresponse transient. Significant problems still remain in managing BCpatients. Notably, failure rates for treating high-grade superficial andinvasive BC remain unacceptably high. In addition, current treatmentsnot only adversely affect patient morbidity, but also present a largeeconomic burden. Newer, more effective therapies that both improvepatient outcomes and are more cost-effective would fill a significantneed.

70-80% of BCs are non-invasive, of which two-thirds initially respond toBacillus Calmette-Guérin (BCG) immunotherapy. The remaining 20-30% areinvasive with high malignant potential and limited options beyondradical cystectomy. Even for non-invasive BC, currently availabletreatments offer limited, transient efficacy: 80% of patients withnon-invasive disease recur and 20-30% progress to potentially lethaldisease. For many of these patients, having relapsed after BCG therapyor been diagnosed with highly invasive tumors, even radical surgery islikely to be ineffective. Overall responses to ‘standard’cisplatin-based combination regimens vary between 39-65%, with 15-25%complete-responders and median survivals up to 16 months. Patients withunresectable metastatic BC also face grim odds with a median survival ofonly 7-20 months and 50% mortality after 5 years. Even followingcystectomy, survival varies from 36-48% at 5 years. Since conventionalchemotherapy, immunotherapy, and surgery have not improved responserates, a pressing unmet medical need exists to develop new approachesthat use different modalities to destroy BC and reduce mortality.

BC represents an attractive target for variant-HSV therapy since i) newapproaches for treating non-invasive and invasive BC are needed; ii) thebladder is a confined reservoir and intravesical instillation ofbiologics such as BCG is an established delivery mode; and iii) clinicaluse of BCG demonstrates that the immune system can be harnessed toattack BC. While both BCG and HSV-based-therapy stimulate anti-tumorimmunity, only HSV oncolytic viruses also directly kill cancer cells andspread through tumor tissue. Thus, variant HSV as describe herein canhave an impact on treating invasive BC, against which BCG isineffective.

Melanoma, such as metastatic melanoma is another target for treatmentwith the oncolytic viruses described herein, e.g., in Example 3, below.Until the approval in 2011 of ipilimumab and zelboraf, no newtherapeutics for the treatment of metastatic melanoma had been approvedfor approximately 20 years. Despite the impressive initial responserates seen in Phase 3 clinical trials for ipilimumab and zelboraf, therates of complete responses are very low for both drugs. Noveltherapeutics such as the oncolytic viruses provided herein are thusneeded.

Novel therapeutics for other cancers, such as, but not limited to,ovarian cancer and glioblastoma are also needed.

Compositions for killing tumor cells and/or for treating cancer in asubject can be advantageously used in combination with other treatmentmodalities, including without limitation radiation, chemotherapy,thermotherapy, molecular targeted therapies, and surgery.

Chemotherapeutic agents used in the methods described herein includewithout limitation taxol, taxotere and other taxoids (e.g., as disclosedin U.S. Pat. Nos. 4,857,653; 4,814,470; 4,924,011, 5,290,957; 5,292,921;5,438,072; 5,587,493; European Patent No. EP 253 738; and PCTPublication Nos. WO 91/17976, WO 93/00928, WO 93/00929, and WO96/01815), cisplatin, carboplatin, (and other platinum intercalatingcompounds), etoposide and etoposide phosphate, bleomycin, mitomycin C,CCNU, doxorubicin, daunorubicin, idarubicin, ifosfamide, methotrexate,mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide,nitrosoureas, mitomycin, dacarbazine, procarbizine, campathecins,dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine,vincristine, vinorelbine, paclitaxel, docetaxel, calicheamicin, and thelike.

Typical radiation therapy includes without limitation radiation at 1-2Gy. Examples of radiation therapy include without limitationγ-radiation, neutron beam radiotherapy, electron beam radiotherapy,proton therapy, brachytherapy, and systemic radioactive isotopes.

Radiation therapy and chemotherapy via local delivery of radioconjugatesand chemotherapeutics, may also be used in the methods described herein.Directing the cytotoxic exposure directly to the tumor itself is acommonly used approach to deliver a cytotoxic drug while minimizing thecytotoxic exposure of normal tissues. However, one of the factors whichlimit the effectiveness of such an approach is incomplete induction oftumor cell death because of limited dose delivery. Thus, it would behighly desirable to concurrently use the variant-HSV containingtherapeutics of the invention to enhance the sensitivity of the tumorcells to the particular cytotoxic agent. Tumor-specific delivery iscommonly achieved by conjugating a cytotoxic agent (e.g., a toxin (suchas ricin) or a radioisotope) to an antibody that preferentially targetsthe tumor (e.g., glypican-3 in hepatocellular carcinoma, anti-CD2 inneuroblastoma, or anti-Her2-neu in certain breast carcinomas. Thetargeting may be also done with natural targeting (i.e., withradioactive iodine in the treatment of thyroid carcinoma), physicaltargeting (i.e., administration of a radioisotope to a particular bodycavity), or other targeting polypeptide (e.g., ferritin inhepatocellular carcinoma).

In addition to combination with conventional cancer therapies such aschemotherapy, radiation therapy, thermotherapy, surgery (tumorresection), TACE (transarterial chemoembolization), variant-HSVoncolytic therapy in tumor or cancer cells can be combined with otheranti-tumor/anti-cancer therapies, including but by no means limited tosmall tyrosine kinase inhibitors (e.g., sorafenib, erlotinib, gefitinib,brivanib, sunitinib, lapatinib, cediranib, vatalanib), monoclonalantibodies (e.g. cetuximab, bevacizumab, IMC-A12, IMC1121B, panitumumab,trastuzumab), suicide gene therapy (i.e., introduction of genes thatencode enzymes capable of conferring to tumor cells sensitivity tochemotherapeutic agents such as thymidine kinase of herpes simplex virusor varicella zoster virus and bacterial cytosine deaminase),anti-oncogene or tumor suppressor gene therapy (e.g., usinganti-oncogene molecules including monoclonal antibodies, single chainantibody vectors, antisense oligonucleotide constructs, ribozymes,immunogenic peptides, etc.), administration of tumor growth inhibitors(e.g., interferon (IFN)-γ, tumor necrosis factor (TNF)-α, TNF-β, andsimilar cytokines, antagonists of tumor growth factor (TGF)-R and IL-10,etc.), administration of angiogenesis inhibitors (e.g., fragments ofangiogenic polypeptides that are inhibitory [such as the ATF ofurokinase], angiogenesis inhibitory factors [such as angiostatin andendostatin], tissue inhibitors of metalloproteinase, soluble receptorsof angiogenic factors [such as the urokinase receptor or FGF/VEGFreceptor], molecules which block endothelial cell growth factorreceptors, and Tie-1 or Tie-2 inhibitors), vasoconstrictive agents(e.g., nitric oxide inhibitors), immune therapies with animmunologically active polypeptide (including immunostimulation, e.g.,in which the active polypeptide is a cytokine, lymphokine, or chemokine[e.g., IL-2, GM-CSF, IL-12, IL-4], and vaccination, in which the activepolypeptide is a tumor specific or tumor associated antigen), and anyother small molecules useful for treating cancer including pro-apoptoticagents (e.g. mapatumumab), proteosome inhibitors (e.g. bortezomib), cellcycle inhibitors (e.g. flavopiridol), DNA methylation inhibitors (e.g.5-Aza-cytidine) and the like.

Tumor load is assessed prior to therapy by means of objective scans ofthe tumor such as with x-ray radiographs, computerized tomography (CATscans), nuclear magnetic resonance (NMR) scans or direct physicalpalpation of the tumor mass. Alternatively, the tumor may secrete amarker substance such as alphafetoprotein from colon cancer, CA 125antigen from ovarian cancer, or serum myeloma “M” protein from multiplemyeloma, or AFP for hepatocellular carcinoma. The levels of thesesecreted products then allow for an estimate of tumor burden to becalculated. These direct and indirect measures of the tumor load aredone pretherapy, and are then repeated at intervals following theadministration of the drug in order to gauge whether or not an objectiveresponse has been obtained. An objective response in cancer therapygenerally indicates >50% shrinkage of the measurable tumor disease (apartial response), or complete disappearance of all measurable disease(a complete response). Typically these responses must be maintained fora certain time period, usually one month, to be classified as a truepartial or complete response. In addition, there may be stabilization ofthe rapid growth of a tumor or there may be tumor shrinkage that is<50%, termed a minor response or stable disease.

In general, increased survival is associated with obtaining a completeresponse to therapy and, in some cases, a partial response if maintainedfor prolonged periods can also contribute to enhanced survival in thepatient. Patients receiving chemotherapy are also typically “staged” asto the extent of their disease before and following chemotherapy arethen restaged to see if this disease extent has changed. In somesituations the tumor may shrink sufficiently and if no metastases arepresent, then surgical excision may be possible after chemotherapytreatment where it was not possible beforehand due to the widespreaddisease. In this case the chemotherapy treatment with the novelpharmaceutical compositions of the invention is being used as anadjuvant to potentially curative surgery. In addition, patients may haveindividual lesions in the spine or elsewhere that produce symptomaticproblems such as pain and these may need to have local radiotherapyapplied. This may be done in addition to the continued use of thesystemic pharmaceutical compositions.

Patients are assessed for toxicity with each course of administration ofa variant HSV of the invention or composition comprising a variant HSV,typically looking at effects on liver function enzymes and renalfunction enzymes such as creatinine clearance or BUN as well as effectson the bone marrow, typically a suppression of granulocytes importantfor fighting infection and/or a suppression of platelets important forhemostasis or stopping blood flow. For such assessments, normal bloodcounts may be reached between 1-3 weeks after therapy. Recovery thenensues over the next 1-2 weeks. Based on the recovery of normal whiteblood counts, treatments may then be resumed.

Typically, complete and partial responses are associated with at least a1-2 log reduction in the number of tumor cells (a 90-99% effectivetherapy), although smaller or larger reductions in tumor burden are alsopossible. Patients with advanced cancer will typically have >10⁹ tumorcells at diagnosis, multiple treatments may be required in order toreduce tumor burden to a very low state and potentially obtain a cure ofthe disease.

At the end of a treatment cycle with a pharmaceutical formulation of theinvention, which could comprise several weeks of continuous drug dosing,patients can be evaluated for response to therapy (complete and partialremissions), toxicity measured by blood work and general well-beingclassified performance status or quality of life analysis. The latterincludes the general activity level of the patient and their ability todo normal daily functions. It has been found to be a strong predictor ofresponse and some anticancer drugs may actually improve performancestatus and a general sense of well-being without causing significanttumor shrinkage. Thus, for some cancers that are not curable, thepharmaceutical formulations may similarly provide a significant benefit,well-being performance status, etc. without affecting true complete orpartial remission of the disease.

A number of biological assays are available to evaluate and to optimizethe choice of variant HSV and compositions comprising variant HSV foroptimal antitumor/anticancer activity. These assays can be roughly splitinto two groups; those involving in vitro exposure of variant HSV totumor/cancer cells and in vivo antitumor/anticancer assays in rodentmodels and rarely, in larger animals.

Cytolytic assays in vitro for variant HSV generally involve the use ofestablished tumor/cancer cell lines both of animal and of human origin.These cell lines can be obtained from commercial sources such as theAmerican Type Tissue Culture Laboratory in Bethesda, Md., and fromtumor/cancer cell banks at research institutions. Exposures to variantHSV may be carried out under simulated physiological conditions oftemperature, oxygen and nutrient availability in the laboratory. Theendpoints for these in vitro assays can involve: 1) colony formation; 2)a simple quantitation of cell division over time; 3) the uptake of socalled “vital” dyes which are excluded from cells with an intactcytoplasmic membrane; 4) the incorporation of radiolabeled nutrientsinto a proliferating (viable) cell. Colony forming assays have been usedboth with established cell lines, as well as fresh tumor biopsiessurgically removed from patients with cancer. In this type of assay,cells are typically grown in petri dishes on soft agar, and the numberof colonies or groups of cells (>60 m in size) are counted eithervisually, or with an automated image analysis system. A comparison isthen made to the untreated control cells allowed to develop coloniesunder identical conditions. Because colony formation is one of thehallmarks of the cancer phenotype, only malignant cells will formcolonies without adherence to a solid matrix. This can therefore be usedas a screening procedure and assay for effectiveness for variant HSV,and there are a number of publications which show that results obtainedin colony forming assays correlate with clinical trial findings with thesame drugs.

The enumeration of the total number of cells is one approach to in vitrotesting with either cell lines or fresh tumor biopsies. In this assay,clumps of cells are typically disaggregated into single units which canthen be counted either manually on a microscopic grid or using anautomated flow system such as either flow cytometry or a Coulter™counter. Control (untreated) cell growth rates are then compared to thetreated (with a nucleic acid) cell growth rates. Vital dye staining isanother one of the older hallmarks of antitumor assays. In this type ofapproach cells either untreated or treated with a cancer drug (e.g.,oncolytic variant HSV), are subsequently exposed to a dye such asmethylene blue, which is normally excluded from intact (viable) cells.The number of cells taking up the dye (dead or dying) is the numeratorwith a denominator being the number of cells which exclude the dye.

In addition to vital dye staining, viability can be assessed using theincorporation of radiolabeled nutrients and/or nucleotides. In tumorcell assays, a typical experiment involves the incorporation of either(3H) tritium- or 14C-labeled nucleotides such as thymidine. Control(untreated) cells are shown to take up a substantial amount of thisnormal DNA building block per unit time, and the rate of incorporationis compared to that in the drug treated cells. This is a rapid andeasily quantifiable assay that has the additional advantage of workingwell for cells that may not form large (countable) colonies. Drawbacksinclude the use of radioisotopes which present handling and disposalconcerns.

There are large banks of human and rodent tumor/cancer cell lines thatare available for these types of assays. Examples of suitable cell linesinclude but are not limited to UMUC3, T24, J82 and EJ (MGH-UI), J82(COT), RT4, RT112, TCCSuP and SCaBER cells, which are bladder cancercell lines. However, cell lines from other types of cancers (e.g., HT29colorectal adenocarcinoma, LNCaP.FGC prostate adenocarcinoma, MDA-MB-231breast adenocarcinoma, SK-MEL-28 malignant melanoma or U-87 MG) are alsosuitable. Other examples of suitable melanoma cell lines include withoutlimitation, A-375, HS-695T, IGR-1, MEL-CLS-1, MEL-CL2, MEL-CLS3,MEL-CLS-4, MEWO, MML01, NIS-G, SK-MEL-, SK-MEL-2 and SK-MEL-5(available, e.g., from Cell Line Services (Germany). Non-limitingexamples of ovarian cancer cell lines, include, e.g., PA-1, Caov-3, SW626 and SK-OV-3. Non-limiting examples of glioblastoma cell linesinclude, e.g., LN-18, U-87 MG, F98, T98G. Such cell lines arecommercially available, e.g., from American Type Culture Collection(ATCC).

The current test system used by the National Cancer Institute uses abank of over 60 established sensitive and multidrug-resistant humancells lines of a variety of cell subtypes. This typically involves 5-6established and well-characterized human tumor/cancer cells of aparticular subtype, such as non-small cell or small cell lung cancer,for testing new agents. Using a graphic analysis system called Compare™,the overall sensitivity in terms of dye uptake (either sulforhodamine Bor MTT tetrazolium dye) is determined. The specific goal of thisapproach is to identify nucleic acids that are uniquely active in asingle histologic subtype of human cancer. In addition, there are a fewsublines of human cancer that demonstrate resistance to multiple agentsand are known to, in some cases, express the multidrug resistance pump,p-glycoprotein. The endpoint for certain assays is the incorporation ofa protein dye called sulforhodamine B (for adherent tumor cells) and thereduction of a tetrazolium (blue) dye in active mitochondrial enzymes(for non-adherent, freely-floating types of cells).

Once a variant HSV of the invention has demonstrated some degree ofactivity in vitro at inhibiting tumor/cancer cell growth and/or atkilling tumor cells, such as colony formation or dye uptake,antitumor/antitumor efficacy experiments are performed in vivo. Rodentsystems can be used for initial assays of antitumor activity since tumorgrowth rates and survival endpoints are well-defined, and since theseanimals generally reflect the same types of toxicity and drug metabolismpatterns as in humans. For this work, syngeneic (same gene line) tumorsare typically harvested from donor animals, disaggregated, counted andthen injected back into syngeneic (same strain) host mice. Variant HSVare typically then injected at some later time point(s), preferably byin situ injection into the tumor site. Tumor growth rates and/orsurvival are determined and compared to untreated controls. In theseassays, growth rates are typically measured for tumors growing in theflank of the animal, wherein perpendicular diameters of tumor width aretranslated into an estimate of total tumor mass or volume. The time toreach a predetermined mass is then compared to the time required forequal tumor growth in the untreated control animals.

In some embodiments, significant findings generally involve a >25%increase in the time to reach the predetermined mass in the treatedanimals compared to the controls. In other embodiments, significantfindings involve a >50% increase in the time to reach the predeterminedmass in the treated animals compared to the controls. The significantfindings are termed “tumor growth inhibition” or “anti-tumor response.”

Human tumors have been successfully transplanted in a variety ofimmunologically deficient mouse models. A mouse called the nu/nu or“nude” mouse can be used to develop in vivo assays of human tumorgrowth. In nude mice, which are typically hairless and lack a functionalthymus gland, human tumors (millions of cells) are typically injected inthe flank and tumor growth occurs slowly thereafter. This visibledevelopment of a palpable tumor mass is called a “take”. Anticancerdrugs such as the variant HSV disclosed herein are then injected by someroute (intravenous, intramuscular, subcutaneous, per os) into or distalto the tumor implant site, and growth rates are calculated byperpendicular measures of the widest tumor widths as described earlier.A number of human tumors are known to successfully “take” in the nudemouse model. An alternative mouse model for this work involves mice witha severe combined immunodeficiency disease (SCID), in which there is adefect in maturation of lymphocytes. Because of this, SCID mice do notproduce functional B- and T-lymphocytes. However, these animals do havenormal natural killer (NK) cell activity. Nonetheless, SCID mice will“take” a large number of human tumors. Tumor measurements and drugdosing are generally performed as above. Again, positive compounds inthe SCID mouse model are those that inhibit tumor growth rate by >20-50%compared to the untreated control.

For in vivo studies, such as for a study for efficacy of a variant HSVof the invention for treating bladder cancer, an orthotopic mouse modelcan be used which closely mimics bladder cancer in humans. The majorutility of orthotopic cancer models is that it allows treatment of atumor within the bladder and intravesical instillation into the bladderto be evaluated as a mode of therapy. Orthotopic models using humantumor cells can be examined in athymic, immunocompromised mice, whereassyngenic murine tumors can be utilized in immune competent mice.Transgenic mice that spontaneously develop tumors in the bladder canalso be used. As disclosed in the Examples, herein, the variant HSV ofthe invention are particularly useful because they are capable ofinhibiting murine TAP, e.g., by the expression of UL49.5 from BHV, andcan thus be studied in immune competent murine models of cancer in whichthe mice are seropositive for HSV-1, and the ability of the improvedvariant HSV of the invention to evade the host immune response, and theimportance of that immune-evasion capability for anti-tumor function ofthe variant HSV can be determined. Such models provide important dataregarding how effective a variant HSV of the invention will be, e.g., inan immune-competent human subject, such as a cancer patient.

The most commonly used immune competent mouse model for evaluatingtherapeutics (such as the variant OV provided herein) for the treatmentof melanoma utilizes mouse B16F10 cells implanted into C57/B16 miceeither s.c. or into organs, such as the brain, in order to initiatetumor formation. The anti-tumor efficacy of the candidate therapeutic isthen evaluated by administration to the animal in any number of ways,including e.g., direct injection into the tumor, injection into themouse vasculature for systemic delivery, or intradermal injection in anarea outside the tumor site. Measurement of tumor size, overall animalsurvival compared to control animals bearing tumors, and induction ofimmune cells that recognize and kill B16F10 cells can be measured asindicators of therapeutic efficacy. The model described in detail inZamarin D, et al. Gene Ther 2009; 16:796-804, which employed B16F10cells to evaluate the in vivo efficacy of an OV for the treatment ofmetastatic melanoma can be used to evaluate the in vivo properties ofthe OV described herein. The mouse model and methods described in TodaM, et al. Hum Gene Ther 1999; 10:385-93, which describes a classicalstudy in the HSV-1 OV field that employed DBA/2 mice harboring bilaterals.c. mouse melanoma tumors derived from cultured M3 melanoma cells canalso be used. The Toda et al. study demonstrated that HSV-1 OV arecapable of eliciting an anti-tumor immune response against melanomacells.

There are a number of mouse ovarian cancer cell lines that are used inimmune competent mice to evaluate the efficacy of therapeutics for thetreatment of metastatic ovarian cancer. Some common mouse ovarian cancercell lines are MOSEC cells, TD8-VEGF, and Dcfb29-VEGF [see, ChalikondaS, et al. Cancer Gene Ther 2008; 15:115-25; Benencia F, et al. CancerBiology & Therapy 2008; 7:1194-205; and Hung C F, et al. Gene Ther 2007;14:20-9]. Metastatic ovarian cancer usually presents as metastatic focilining the peritoneal cavity. Therefore, most models involveintraperitoneal injection of cultured mouse ovarian cancer cells inorder to establish metastatic ovarian cancer lesions lining theperitoneal cavity. OV, e.g., the recombinant OV described herein, canthen be instilled into the peritoneal cavity to facilitate infection ofall tumors accessible to the virus. As with bladder and melanoma models,OV therapeutic efficacy can be measured by monitoring tumor size overtime and overall animal survival compared to control animals bearingtumors, as well as induction of immune cells that recognize and kill thecancer cells.

4C8 and 203GL mouse glioblastoma cell lines can be used in immunecompetent mice to evaluate the efficacy of therapeutics for thetreatment of glioblastoma [see, Hellums E K, et al. Neuro-oncology 2005;7:213-24; Markert J M, et al. J Virol 2012; 86:5304-13; and Todo T, etal. Hum Gene Ther 1999; 10:2741-55]. Mouse glioblastoma modelstypically, although not necessarily, employ orthotopic tumorsestablished by drilling a burr hole through the mouse cranium, theninjecting cultured mouse glioma cells into the frontal lobes and closingthe wound with a suture. At a predetermined time point afterintracranial tumor implantation, the burr holes are reopened and OV aredirectly injected into the tumor. Overall animal survival compared tocontrol animals bearing tumors can be used as a measure of the efficacyof the therapy, since tumor size can typically only be measuredpost-mortem. Examples of murine models of glioma are described, e.g., inBruggeman et al. 2007; Cancer Cell; 12(4):328-341; and Marumoto T, etal. Nat Med. 2009 15(1):110-6.

All of these test systems are generally combined in a serial order,moving from in vitro to in vivo, to characterize the antitumor activityof an oncolytic variant HSV of the invention. In general, one wishes tofind out what tumor types are particularly sensitive to a variant HSVand conversely what tumor types are intrinsically resistant (e.g.,non-permissive) to a variant HSV in vitro. Using this information,experiments are then planned in rodent models to evaluate whether or notthe variant HSV that have shown activity in vitro will be tolerated andactive in animals. The initial experiments in animals generally involvetoxicity testing to determine a tolerable dose schedule and then usingthat dose schedule, to evaluate antitumor efficacy as described above.Active variant HSV from these two types of assays may then be tested inhuman tumors growing in SCID or nude mice and if activity is confirmed,these variant HSV then become candidates for potential clinical drugdevelopment.

Administration

The variant HSV of the invention or compositions, e.g., pharmaceuticalcompositions, comprising the variant HSV, may be administered to anindividual, e.g., patient, preferably a human patient, in need oftreatment. A subject or patient in need of treatment is an individualsuffering from cancer, preferably an individual with a solid tumor, andpreferably is one who would benefit by the administration of the variantHSV or pharmaceutical composition thereof. The aim of therapeutictreatment is to improve the condition of a patient. Typically, althoughnot necessarily, therapeutic treatment using a variant HSV orpharmaceutical composition of the invention alleviates the symptoms ofthe cancer. A method of treatment of cancer according to the inventioncomprises administering a therapeutically effective amount of a variantHSV of the invention or of a pharmaceutical composition containing thevariant HSV to a patient suffering from cancer. Administration of anoncolytic variant HSV or composition of the invention to an individualsuffering from a tumor will typically kill the cells of the tumor, thusdecreasing the size of the tumor and/or reducing or preventing spread ofmalignant cells from the tumor.

A variant HSV or pharmaceutical composition thereof can be introducedparenterally, transmucosally, e.g., orally (per os), nasally, orrectally, or transdermally. Parental routes include intravenous,intra-arteriole, intra-muscular, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial administration. Forexample, a variant HSV-containing composition can be administered byinjection, infusion, instillation or inhalation. A preferred route ofadministration is by direct injection. For example, therapeutictreatment may be carried out following direct injection of the variantHSV composition into target tissue (i.e., “in situ administration”). Thetarget tissue may be the tumor or a blood vessel supplying the tumor.

A variant HSV-containing compositions may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents.

In addition to the formulations described previously, variantHSV-containing compositions may also be formulated as a depotpreparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the variant HSV-containingcompositions may be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. In yet another embodiment, the therapeuticcompound can be delivered in a controlled release system. For example, avariant HSV may be administered using intravenous infusion with acontinuous pump, in a polymer matrix such as poly-lactic/glutamic acid(PLGA), a pellet containing a mixture of cholesterol and the activeingredient (SilasticR™; Dow Corning, Midland, Mich.; see U.S. Pat. No.5,554,601) implanted subcutaneously, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. Inanother embodiment, the active ingredient can be delivered in a vesicle,in particular a liposome (see Langer, Science 249:1527-1533 (1990);Treat et al., in Liposomes in the Therapy of Infectious Disease andCancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp. 353-365(1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

The routes of administration and dosages described are intended only asa guide since a skilled practitioner will be able to determine readilythe optimum route of administration and dosage. The dosage may bedetermined according to various parameters, especially according to thelocation of the tumor, the size of the tumor, the age, weight andcondition of the patient to be treated and the route of administration.Preferably the virus is administered by direct injection into the tumor.The virus may also be administered systemically or by injection into ablood vessel supplying the tumor. The optimum route of administrationwill depend on the location and size of the tumor.

Administration of a variant HSV-containing composition may be once aday, twice a day, or more often, but frequency may be decreased during amaintenance phase of the disease or disorder, e.g., once every second orthird day instead of every day or twice a day. The dose and theadministration frequency will depend on the clinical signs, whichconfirm maintenance of the remission phase, with the reduction orabsence of at least one or more preferably more than one clinical signsof the acute phase known to the person skilled in the art. Moregenerally, dose and frequency will depend in part on recession ofpathological signs and clinical and subclinical symptoms of a diseasecondition or disorder contemplated for treatment with the presentcompounds.

Keeping the above description in mind, the amount of virus administeredin the case of HSV can be in the range of from 10⁴ to 10¹⁰ pfu,preferably from 10⁵ to 10⁸ pfu, more preferably about 10⁶ to 10⁹ pfu.Typically 1-4 ml, such as 2 to 3 ml of a pharmaceutical compositionconsisting essentially of the virus and a pharmaceutically acceptablesuitable carrier or diluent would be used for direct injection into anindividual tumor. [See, Senzer et al. J Clin Oncol (2009)27(34):5763-5771.] However for some oncolytic therapy applicationslarger volumes up to 10 ml may also be used, depending on the tumortype, tumor size and the inoculation site. Likewise, smaller volumes ofless than 1 ml may also be used. Dosages and administration regimen canbe adjusted depending on the age, sex and physical condition of thesubject or patient as well as the benefit of the treatment and sideeffects in the patient or mammalian subject to be treated and thejudgment of the physician, as is appreciated by those skilled in theart.

The present invention is described here by means of the followingexamples. However, the use of examples anywhere in the specification isillustrative of and in no way limits the scope and meaning of theinvention or of any exemplified terms. Likewise, the invention is notlimited to any particular embodiment described herein. Indeed, manymodifications and variations to those skilled in the art upon readingthis specification and can be made without departing from its spirit andscope. The invention is therefore to be limited only by the terms of theappended claims along with the full scope of equivalents to which theclaims are entitled. The disclosures of all citations, including issuedpatents, published applications, and scientific articles, in thespecification are expressly incorporated herein by reference in theirentirety.

It is to be understood that numerical values of binding activities andother parameters reported in the examples, and throughout the entirespecification, are approximate. Individual measurements of theseparameters may vary, e.g., due to normal experimental error and/ordepending on the specific conditions used.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition. Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989 (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); Ausubel, F. M. et al.(eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc.,1994. These techniques include site directed mutagenesis as described inKunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985), U.S. Pat. No.5,071,743, Fukuoka et al., Biochem. Biophys. Res. Commun. 263: 357-360(1999); Kim and Maas, BioTech. 28: 196-198 (2000); Parikh andGuengerich, BioTech. 24: 4 28-431 (1998); Ray and Nickoloff, BioTech.13: 342-346 (1992); Wang et al., BioTech. 19: 556-559 (1995); Wang andMalcolm, BioTech. 26: 680-682 (1999); Xu and Gong, BioTech. 26: 639-641(1999), U.S. Pat. Nos. 5,789,166 and 5,932,419, Hogrefe, Strategies 14.3: 74-75 (2001), U.S. Pat. Nos. 5,702,931, 5,780,270, and 6,242,222,Angag and Schutz, Biotech. 30: 486-488 (2001), Wang and Wilkinson,Biotech. 29: 976-978 (2000), Kang et al., Biotech. 20: 44-46 (1996),Ogel and McPherson, Protein Engineer. 5: 467-468 (1992), Kirsch andJoly, Nuc. Acids. Res. 26: 1848-1850 (1998), Rhem and Hancock, J.Bacteriol. 178: 3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28:197-198 (1995), Barrenttino et al., Nuc. Acids. Res. 22: 541-542 (1993),Tessier and Thomas, Meths. Molec. Biol. 57: 229-237, and Pons et al.,Meth. Molec. Biol. 67: 209-218.

EXAMPLES Example 1: Genetic Properties of HSV-1 and Oncolytic StrainsThereof

This Example describes the genetic construction of a neuro-attenuatedvariant HSV (strain Patton) having intact endogenous U_(S)112 andU_(S)11, in which the γ₁34.5 genes are replaced with U_(S)11 fused to animmediate early (IE) promoter,

The HSV-1 genome comprises two unique genome segments, referred to asthe Unique-long (U_(L)) and Unique-short (U_(S)) segments. Both theU_(L) and U_(S) sequences are flanked by inverted terminal repeats,illustrated as empty rectangles in FIGS. 1A-1C. In the wild-type HSV(FIG. 1A) the γ₁34.5 gene, which confers neurovirulence, is a diploidelement located within the inverted repeats flanking the U_(L) segment.The location and arrangement of the U_(S)11 and U_(S)12 genes areindicated and expanded below the HSV-1 genome in FIG. 1A. The U_(S)12gene is expressed very early during infection by an immediate earlypromoter (denoted by star-12 in FIG. 1A). The U_(S)11 gene is expressedlate in viral infection by a separate promoter (denoted by star-11 inFIG. 1A) that is contained within the U_(S)12 gene.

In the modified HSV-1 OncoVex^(GMCSF) (FIG. 1B), the γ₁34.5 genes arereplaced by the CMV promoter, fused to the gene encoding humanGranulocyte-Macrophage Colony Stimulating Factor or “GM-CSF”. Deletionof γ₁34.5 results in neuro-attenuation, but it also results in a severereduction in the ability of the virus to overcome a cellular block toviral replication during infection of many cancer cell lines. Toovercome this deficiency, U_(S)12 is deleted in order to directsynthesis of U_(S)11 from the immediate early U_(S)12 promoter,resulting in U_(S)11 accumulation prior to the crippling proteinsynthesis block. However, while this maximizes protein synthesis duringinfection, loss of U_(S)12 results in a viral inability to evade CD8+cytolytic T-cell killing of infected cells, leading to enhanced viralclearance, decreased cell killing by the virus, and reduced overallsynthesis of GM-CSF.

In order to address the deficiencies in OncoVEX^(GMCSF), aneuro-attenuated variant HSV (strain Patton) having intact endogenousUs12 and Us11, in which the γ₁34.5 genes are replaced with Us11 fused toan immediate early (IE) promoter, was generated, as described in detailin U.S. Pat. No. 7,731,952.

To generate an avirulent Δ34.5 virus that expresses Us11 at immediateearly (IE) times and preserves the immunomodulatory Us12 gene, both theγ₁34.5 promoter and ORF of the HSV-1 genome were replaced by cloning theUs11 gene, under transcriptional control of the α27 IE promoter, betweenthe DraI and SacI sites of Bam SP, as shown in FIG. 1C. This fragmentwas cotransfected into Vero cells with purified Δ34.5 virus DNA andrecombinants were selected on U251 glioblastoma cells which arenon-permissive for the growth of Δ34.5 viruses that do not express Us11at IE times. This modified virus was named: Δ34.5::flα27P-Us11(“OV-2711”). FIG. 1C is a detailed map of the genome of OV-2711,including restriction sites, and shows the location of the Us11 genesthat have replaced the two WT γ₁34.5 genes, while leaving intact theUs11 and Us12 loci. Thus, the modified OV-2711 variant HSV, which is thebasis for the novel, improved variant HSV of the present invention, hasthree functional Us1 genes, as shown in the simplified line diagram inFIG. 1C.

The OV-2711 construct directs synthesis of Us11 throughout the entireviral lifecycle, from both the ectopic IE promoter as well as theendogenous late promoter (denoted by star-11 in FIG. 1), located withinthe Us12 gene, leading to better viral yields and improved oncolysis.

Example 2: Oncolytic Variant HSV Optimized for Immune-Competent MurineModel

This Example describes improved variant HSV that can be generated basedon the OV-2711 HSV described in Example 1, and that can be tested in animmune-competent murine model of cancer.

IE-Us11 is a very powerful dominant selectable marker when inserted intoΔ34.5 HSV-1. To isolate a variant HSV containing IE-Us11, the Us11 geneis fused to the HSV-1 IE promoter α27 and this expression cassette isinserted into a targeting vector in place of γ₁34.5 (see, U.S. Pat. No.7,731,952). Specifically, the targeting vector is the viral BamSPfragment cloned into plasmid pBR322 (Invitrogen, Carlsbad, Calif.). TheIE-Us11 cassette is cloned into BamSP between the DraI site upstream ofthe 34.5 ORF and the second SacI site, downstream of the 34.5 ORF, inorder to replace the 34.5 ORF. Flanking the α27-Us11 expression cassetteare sequences that mediate homologous recombination into the γ₁34.5locus. Specifically, a SacI-BamHI fragment that is downstream of the34.5 ORF and the DraI-BamH1 fragment that is upstream of the 34.5 ORF inthe BamSP fragment.

There are two SacI-BamHI fragments in the HSV locus. The firstSacI-BamHI fragment has the following sequence in the HSV-1 strain 17sequence (GenBank Accession No. X14112 (SEQ ID NO: 1)), occurring atnucleotides 1307-2910:

(SEQ ID NO: 12) ccgcaccaagccgctctccggagagacgatggcaggagccgcgcatatatacgcttggagccagcccgccctcacagggcgggccgcctcgggggcgggactggccaatcggcggccgccagcgcggcggggcccggccaaccagcgtccgccgagtcttcggggcccggcccattgggcgggagttaccgcccaatgggccgggccgcccacttcccggtatggtaattaaaaacttgcaagaggccttgttccgcttcccggtatggtaattagaaactcattaatgggcggccccggccgcccttcccgcttccggcaattcccgcggccataatgggcaaccccggtattccccgcctcccgcgccgcgcgtaaccactcccctggggaccgggttatgctaattgcttttttggcggaacacacggcccctcgcgcattggcccgcgggtcgctcaatgaacccgcattggtcccctggggttccgggtatggtaatgagtttcttcgggaaggcgggaagccccggggcaccgacgcaggccaagcccctgttgcgtcggcgggaggggcatgctaatggggttctttgggggacaccgggttgggcccccaaatcgggggccgggccgtgcatgctaatgatattctttgggggcgccgggttggtccccggggacggggccgccccgcggtgggcctgcctcccctgggacgcgcggccattgggggaatcgtcactgccgcccctttggggaggggaaaggcgtggggtataagttagccctggcccgacagtctggtcgcatttgcacctcggcactcggagcgagacgcagcagccaggcagactcgggccgccccctctccgcatcaccacagaagccccgcctacgttgcgacccccagggaccctccgtccgcgaccctccagccgcatacgacccccatggagccccgccccggagcgagtacccgccggcctgagggccgcccccagcgcgaggtgaggggccgggcgccatgtctggggcgccatattggggggcgccatattggggggcgccatgagggggacccccgacccttacactggaaccggccgccatgttgggggaccccatcatacacgggagccgggcgccatgttggggcgccatgttagggggcgtggaaccccgtgacactatatatacagggaccgggggcgccatgttagggggtgcggaaccccctgaccctatatatacagggaccggggtcgccctgagggggtcgccatgtgaccccctgactttatatatacagacccccaacacatacacatggcccctttgactcagacgcagggcccggggtcgccgtgggaccccctgactcatacacagagacacgcccccacaacaaacacacaaggaccggggtcgccgtgttgggggcgtggtccccactgactcatacgcaggccccccttactcacacgcatctaggggggtggggaggagccgcccgccatatttgggggacgccgtgggacccccgactccggtgcgtctggagggcgggagaagagggaagaagaggggtcgggatcc.

The second SacI-BamHI fragment has the following sequence in the HSV-1strain 17 sequence (GenBank Accession No. X14112) (SEQ ID NO: 1)),occurring at nucleotides 123461-125064:

(SEQ ID NO: 13) ggatcccgacccctcttcttccctcttctcccgccctccagacgcaccggagtcgggggtcccacggcgtcccccaaatatggcgggcggctcctccccacccccctagatgcgtgtgagtaaggggggcctgcgtatgagtcagtggggaccacgcccccaacacggcgaccccggtccttgtgtgtttgttgtgggggcgtgtctctgtgtatgagtcagggggtcccacggcgaccccgggccctgcgtctgagtcaaaggggccatgtgtatgtgttgggggtctgtatatataaagtcagggggtcacatggcgacccccaacagggcgaccccggtccctgtatatatagggtcagggggttccgcaccccctaacatggcgcccccggtccctgtatatatagtgtcacggggttccacgccccctaacatggcgccccaacatggcgcccggctcccgtgtatgagtgggggtcccccaacatggcggccggttccagtgtaagggtcgggggtcccccaacatggcgccccccaatatggcgccccccaatatggcgccccagacatggcgcccggcccctcacctcgcgctgggggcggccctcaggccggcgggtactcgctccggggcggggctccatgggggtcgtatgcggctggagggtcgcggacggagggtccctgggggtcgcaacgtaggcggggcttctgtggtgatgcggagagggggcggcccgagtctgcctggctgctgcgtctcgctccgagtgccgaggtgcaaatgcgaccagactgtcgggccagggctaacttataccccacgccaccccctccccaaaggggcggcagtgacgattcccccaatggccgcgcgtcccaggggaggcaggcccaccgcggggcggccccgtccccggggaccaacccggcgcccccaaagaatatcattagcatgcacggcccggcccccgatttgggggcccaacccggtgtcccccaaagaaccccattagcatgcccctcccgccgacgcaacaggggcttggcctgcgtcggtgccccggggcttcccgccacccgaagaaactcattaccatacccggaaccccaggggaccaatgcgggttcattgagcgacccgcgggccaatgcgcgaggggccgtgtgttccgccaaaaaagcaattagcataacccggaaccccaggggagtggttacgcgcggcgcgggaggcggggaataccggggttgcccattaagggccgcgggaattgccggaagcgggaagggcggccggggccgcccattaatgagtttctaattaccataccgggaagcggaacaaggcctcttgcaagtttttaattaccataccgggaagtgggcggcccggcccattgggcggtaactcccgcccaatgggccgggccccgaagactcggcggacgctggttggccgggccccgccgcgctggcggccgccgattggccagtcccgcccccgaggcggcccgccctgtgagggcgggctggctccaagcgtatatatgcgcggctcctgccatcgtctctccggagagcggcttggtgc gg.

The DraI-BamHI fragment, having a single location in HSV, has thefollowing sequence in the HSV-1 strain 17 sequence (GenBank AccessionNo. X14112 (SEQ ID NO: 1)), found in nucleotides 125990-129564:

(SEQ ID NO: 14) aaagggccgcgcgcgacccccggggggtgtgtatggggggggcccgtatcggcgtctggccgctcctccccccgctcctccccccgctcctccccccgctcctccccccgctcaccccccgctcctccccccgctcctccccccgctcctccccccgacctccccccgctcctccccccgctcctccccccgctcctccccccgctcctccccccgctcctccccccgctcctccccccgctcctccccccgctcctccccccgctcctccccccgctcccgcggccccgccccccacgcccgccgcgcgcgcgcacgccgcccggaccgccgcccgccttttttgcgcgcgcgcgcgcccgcggggggcccgggctgccacaggtgaaaccaacagagcacggcgcactccgcacgtcacacgtcacgtcatccaccacacctgcccaacaacacaactcacagcgacaactcaccgcgcaacaactcctgttcctcatccacacgtcaccgcgcacctcccgctcctccagacgtaccccggcgcaacacaccgctcctgctacacaccaccgccccctccccagccccagccctccccagccccagccctccccggccccagccctccccggccccagccctccccggccccagccctccccggccccagccctccccggccccagccctccccggccccagccctccccggcgcgtcccgcgctccctcgggggggttcgggcatctctacctcagtgccgccaatctcaggtcagagatccaaaccctccgggggcgcccgcgcaccaccaccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgccccctcccgcccctcgaataaacaacgctactgcaaaacttaatcaggttgttgccgtttattgcgtcttcgggtctcacaagcgccccgccccgtcccggcccgttacagcaccccgtccccctcgaacgcgccgccgtcgtcttcgtcccaggcgccttcccagtccacaacttcccgccgcgggggcgtggccaagcccgcctccgcccccagcacctccacggcccccgccgccgccagcacggtgccgctgcggcccgtggccgaggcccagcgaatcccgggcggcgccggcggcagggcccccgggccgtcgtcgtcgccgcgcagcaccagcgggggggcgtcgtcgtcgggctccagcagggcgcgggcgcaaaagtccctccgcggcccgcgccaccgggccgggccggcgcgcaccgcctcgcgccccagcgccacgtacacgggccgcagcggcgcgcccaggccccagcgcgcgcaggcggcgtgcgagtgggcctcctcctcgcagaagtccggcgcgccgggcgccatggcgtcggtggtccccgaggccgccgcccggccgtccagcgccggcagcacggcccggcggtactcgcgcggggacatgggcaccggcgtgtccgggccgaagcgcgtgcgcacgcggtagcgcacgttgccgccgcggcacaggcgcagcggcggcgcgtcggggtacaggcgcgcgtgcgcggcctccacgcgcgcgaagacccccgggccgaacacgcggcccgaggccagcaccgtgcggcgcaggtcccgcgccgccggccagcgcacggcgcactgcacggcgggcagcagctcgcacgccaggtaggcgtgctgccgcgacaccgcgggcccgtcggcgggccagtcgcaggcgcgcacggtgttgaccacgatgagccgccggtcgccggcgctggcgagcagccccagaaactccacggccccggcgaaggccaggtcccgcgtggacagcagcagcacgccctgtgcgcccagcgccgacacgtcgggggcgccggtccaattgcccgcccaggcggccgtgtccggcccgcacagccggttggccagggccgccagcaggcaggacagcccgccgcgctcggcggaccactccggcggcccccccgaggccccgccgccggccaggtcctcgcccggcagcggcgagtacagcaccaccacgcgcacgtcctcggggtcggggatctggcgcatccaggccgccatgcggcgcagcgggcccgaggcgcgcagggggccaaagaggcggcccccggcggccccgtgggggtgggggttatcgtcgtcgtcgccgccgccgcacgcggcctgggcggcgggggcgggcccggcgcaccgcgcggcgatcgaggccagggcccgcgggtcaaacatgagggccggtcgccaggggacggggaacagcgggtggtccgtgagctcggccacggcgcgcggggagcagtaggcctccagggcggcggccgcgggcgccgccgtgtggctgggccccgggggctgccgccgccagccgcccagggggtcggggccctcggcgggccggcgcgacacggccacggggcgcgggcgggcctgcgccgcggcggcccggggcgccgcgggctgggcgggggcgggctcgggccccgggggcgtggaggggggcgcgggcgcggggaggggggcgcgggcgtccgagccgggggcgtccgcgccgctcttcttcgtcttcgggggtcgcgggccgccgcctccgggcggccgggccgggccgggactcttgcgcttgcgcccctcccgcggcgcggcggaggcggcggcggccgccagcgcgtcggcggcgtccggtgcgctggccgccgccgccagcagggggcgcaggctctggttgtcaaacagcaggtccgcggcggcggcggccgcggagctcggcaggcgcgggtcccgcggcagcgcggggcccagggccccggcgaccaggctcacggcgcgcacggcggccacggcggcctcgctgccgccggccacgcgcaggtccccgcgcaggcgcatgagcaccagcgcgtcgcgcacgaaccgcagctcgcgcagccacgcgcgcaggcggggcgcgtcggcgtgcggcggcggcggggaagcggggcccgcgggtccctccggccgcggggggctggcgggccgggccccggccagccccgggacggccgccaggtcgccgtcgaagccctcggccagcgcctccaggatcccgcggcaggcggccaggcactcgacggccacgcggccggcctgggcgcggcgcccggcgtcgtcgtcggcgtcggcgtggcgggcggcgtcggggtcgtcgccccccgcgggggaggcgggcgcggcggacagccgccccagggcggcgaggatcc.

This vector is then co-transfected with purified Δ34.5 viral DNA (asdescribed in U.S. Pat. No. 7,731,952) into Vero cells (ATCC No. CCL-81),and IE-Us11 expressing viruses are selected by two passages on U251 cellmonolayers. U251 cells [Ren, Y. et al. (2010) BMC Cancer 10:27] impose apotent translational block on Δ34.5 viruses, but IE-Us11 expressionovercomes this block to viral growth and recombinant IE-Us11 expressingviruses are thus easily selected. Variant HSV are created by insertingexpression cassettes encoding UL49.5 and/or murine GM-CSF (“mGM-CSF”)under either the CMV or EF1α promoters adjacent to the α27-Us11 dominantselectable marker present in the γ₁34.5 locus targeting vector. Theinsertion of mGM-CSF into the γ₁34.5 locus in variant HSV based onOV-2711 (having intact endogenous Us111 and Us112 genes and lackingfunctional ICP34.5 expression) is expected to create a significantlybetter biologic for cancer treatment. These constructs are thenco-transfected with purified Δ34.5 viral DNA into Vero cells.Recombinants are then selected by passage on U251 cells and plaquepurified. Table 1, below, lists examples of the variant HSV that may becreated using this robust selection mechanism.

TABLE 1 OV-2711 Variants Functions Encoded in Virus Virus Name Us12α27-Us11 UL49.5 mGMCSF OV-2711 + + OV-UL49.5 + + + OV-UL49.5-fs + +OV-mGMCSF + + + OV-UL49.5/mGMCSF + + + +

The generation of constructs and isolation of viruses encodingconstructs with the functions indicated in Table 1 are described indetail in Example 3, below.

To obtain genetic configurations that allow effective viral growth andsynthesis of ectopic proteins, the targeting vector constructionstrategy illustrated in FIG. 2 is executed. The γ₁34.5 locus targetingvector is depicted at the top of the figure. This vector is derived fromthe viral BamSP fragment, and γ₁34.5, located between the DraI and SacIsites of BamSP, is replaced by the α27-Us11 dominant selectable marker.In this process, the SacI site is destroyed and the DraI site replacedby a PacI site.

CMV and EF1α promoter cassettes expressing either UL49.5 or GM-CSFflanked by the indicated restriction endonuclease sites (selected fromPacI, Sal-I, XhoI, SacI, DraI, BamHI, BlpI) are synthesized de novo(available as a commercial service, e.g., from the contract manufacturerGenScript USA). CMV-based cassettes are terminated by the BGHpolyadenylation signal and EF1α terminated by the SV40 latepolyadenylation signal. All synthesized cassettes can be inserted intothe targeting vector by digestion with PacI followed by ligation andtransformation into E. coli. By utilizing one restriction enzyme site,expression cassette insertions in both orientations can be constructedsimultaneously. In addition, the placement of the Sal-I and XhoI sitesin the synthesized cassettes facilitates facile construction of CMV andEF1α cassette combinations in all possible orientations relative to eachother (Sal-I and XhoI have compatible cohesive ends). These combinationconstructs are then inserted into the PacI site of the targeting vectorin both orientations. Execution of this strategy generates 22 targetingconstructs, which are shown in FIGS. 3A-3D. As shown in FIGS. 3A-3D, thevarious transcription cassettes are oriented either in the samedirection or run into each other (i.e., have opposite orientations). Thelast two cassettes shown at the bottom of FIG. 3D illustrate aback-to-back orientation of the CMV and EF1α promoters if partialdigestion of a CMV cassette with Sal-I is performed and an XhoI digestedEF1α cassette is inserted into the Sal-I site 3′ of the CMV promoter.The same construct may be made by partially digesting EF1α cassette withXhoI and inserting a Sal-I digested CMV cassette into the XhoI site 3′of the EF1α promoter.

Internal to α27 promoter fragment in the targeting vector is a copy ofthe HSV-1 gK gene. The location of the gK gene and the proposed locationof the gK promoter are shown in the targeting construct illustrated atthe top of FIG. 3A.

An exemplary nucleic acid sequence of the gK gene, is:

(SEQ ID NO: 15) atgctcgccgtccgttccctgcagcacctctcaaccgtcgtcttgataacggcgtacggcctcgtgctcgtgtggtacaccgtcttcggtgccagtccgctgcaccgatgtatttacgcggtacgccccaccggcaccaacaacgacaccgccctcgtgtggatgaaaatgaaccagaccctattgtactgggggccccgacgcacccccccaacgggggctggcgcaaccacgcccatatctgctacgccaatcttatcgcgggtagggtcgtgcccttccaggtcccacctgacgccatgaatcgtcggatcatgaacgtccacgaggcagttaactgtctggagaccctatggtacacacgggtgcgtctggtggtcgtagggtggttcctgtatctggcgttcgtcgccctccaccaacgccgatgtatgtttggcgtcgtgagtcccgcccacaagatggtggccccggccacctacctcttgaactacgcaggccgcatcgtatcgagcgtgttcctgcagtacccctacacgaaaattacccgcctgactgcgagctgtcggtccagcggcaaaacctggttcagttgtttgagacggacccggtcaccttcttgtaccaccgccccgccatcggggtcatcgtaggctgcgagttgatgctacgattgtggccgtgggtctcatcgtcggcaccgattcatatcccggggggcatgtgcgatcacataccccctgtactgaccatcaccacctggtgattgtctccaccatcggcctgacagagctgtattgtattctgcggcggggcccggcccccaagaacgcagacaaggccgccgccccggggcgatccaaggggctgtcgggcgtctgcgggcgctgctgttccatcatcctctcgggcatcgcagtgcgattgtgttatatcgccgtggtggccggggtggtgctcgtggcgcttcactacgagcaggagatccagaggcgcctgt ttgatgtatga.

The ATG initiation codon for gK lies approximately 200 bp downstreamfrom the PacI site and is oriented towards Us11. gK is polyadenylated ata polyA signal located upstream of the α27 promoter transcriptioninitiation site. Insertion of gK into the γ₁34.5 locus results in thecreation of two additional copies for a total of three gK genes. Incertain embodiments, an expression cassette inserted into the PacI sitedoes not interfere with gK expression.

To determine which promoter combinations and orientations yield the bestisolates for the viral panel created using the targeting constructsshown in FIGS. 3A-3D, targeting constructs are linearized andco-transformed with purified Δ34.5 viral DNA into Vero cells. The methodis described in U.S. Pat. No. 7,731,952. Once viral cytopathic effect(cpe) is observed, the plates are freeze-thawed, viral lysatessonicated, diluted 1:10 and 1:100 and added to confluent monolayers ofU251 cells. Once 90% cpe is observed, a second round of U251 selectionis performed using 1:100 and 1:1000 dilutions of viral lysate. When 90%cpe is observed at the lowest dilution, plates are freeze-thawed andviral titers determined.

High titer stocks are then prepared by infection of 10 cm dishescontaining confluent Vero cell monolayers at a MOI of 0.01. These stocksare then titered and used to infect confluent monolayers of U251 cellsat MOI=5. At 16 hrs post-infection, supernatants are removed and storedfor ELISA to determine the level of secreted mGM-CSF. Minimal mediumcontaining radioactive cysteine and methionine is then added to labelnewly synthesized proteins.

The cell monolayers are then lysed in laemmli's buffer and separated bySDS-PAGE on duplicate gels. One gel is processed for Western blotanalysis to detect transgene (e.g., UL49.5 and/or GM-CSF) expression andthe other is fixed, dried and exposed to X-ray film to determine therelative rates of viral protein synthesis at 15 hours post-infection.Pools of OV-2711 variants that replicate to high titers, maintain robustrates of viral protein synthesis and express UL49.5 or GM-CSF or bothare plaque purified and high titer stocks prepared.

Two (2) 10 cm dishes of confluent Vero cell monolayers are then infectedat high MOI for each of three isolates per virus listed in Table 1 andfor which the gene expression cassettes are shown in FIGS. 2 and 3.Viral DNA is then isolated and analyzed by Southern blot to verifyproper integration of the targeting vectors into the γ₁34.5 locus andmaintenance of the Us12 gene. Variant HSV that grow to similar titersand efficiently express either mGM-CSF or UL49.5 or both transgenes(where each transgene is expressed at levels similar to the variant HSVwith one copy of the corresponding transgene) are generated. The variantHSV generated in this Example encode Us12, however, they are deficientin CD8+ T-cell evasion in mice unless they express UL49.5 because Us12does not inhibit rodent TAP.

Example 3: Generation and Isolation of Recombinant Viruses EncodingUL49.5 and GM-CSF

This Example describes the generation and isolation of variants of thewild-type HSV Strain Patton based on the OV-2711 constructs described inExample 2, above, encoding diploid CMV or EF1α promoter-controlledmurine GM-CSF (mGM-CSF) gene and bovine herpes virus (BHV) UL49.5 genealone and together.

The strategy for making the targeting vector constructs illustrated inFIG. 4 was based on linking ectopic transcription units to the immediateearly expressed Us11 (IE-Us11) cassette in a vector that targetshomologous recombination to the viral γ34.5 locus, as described inExample 2, above. The strategy is illustrated in FIG. 5.

The target vector construction strategy proceeded in three steps, asfollows:

Step 1: pCMV-mGM-CSF was digested with NheI and XhoI to release theGM-CSF gene and the vector backbone was gel purified away from theGM-CSF containing fragment. The NheI-XhoI fragments from pNhe-UL49.5-Xhoand pNhe-UL49.5-fs-Xho were isolated by digestion with NheI and XhoIfollowed by gel purification. These fragments were then ligated intoNheI-XhoI digested pCMV-mGM-CSF to create pCMV-UL49.5 andpCMV-UL49.5-fs, respectively.

Step 2: pCMV-mGM-CSF was digested with BamHI and XbaI and the fragmentcontaining the mGM-CSF gene under CMV promoter and BGH polyA control aswell as the entire vector backbone necessary for replication,segregation and maintenance in Escherichia coli was gel purified.pEF1α-UL49.5 was then digested with BamHI and XbaI and the fragmentcontaining the UL49.5 gene under EF1α and SV40 late polyA control wasgel purified and ligated into XbaI-BamHI cut pCMV-GM-CSF to createpCMV-mGM-CSF/EF1α-UL49.5.

Step 3: pCMV-mGM-CSF, pCMV-UL49.5, pCMV-UL49.5-fs, andpCMV-mGM-CSF/EF1α-UL49.5 were digested with BlpI and PacI and thefragments containing the mGM-CSF and/or UL49.5 expression cassettes weregel purified and ligated into BlpI and PacI cut and purified vectorpSP-Δ34.5-fla27P-Us1-PacI to create the targeting vectors:pSP-Δ34.5-fla27P-Us11-CMV-mGM-CSF (having the nucleic acid sequence setforth in SEQ ID NO: 16), pSP-Δ34.5-fla27P-Us11-CMV-UL49.5 (having thenucleic acid sequence set forth in SEQ ID NO: 17),pSP-Δ34.5-fla27P-Us11-CMV-UL49.5-fs (having the nucleic acid sequenceset forth in SEQ ID NO: 18),pSP-Δ34.5-fla27P-Us11-CMV-mGM-CSF/EF1α-UL49.5 (having the nucleic acidsequence set forth in SEQ ID NO: 19), andpSP-Δ34.5-fla27P-Us11-CMV-mGM-CSF/EF1α-UL49.5-fs (having the nucleicacid sequence set forth in SEQ ID NO: 20). Upon successful homologousrecombination, the variant HSV comprise the polynucleotide cassetteswithout the flanking homologous recombination regions. Thus the variantHSV comprise polynucleotide cassettes that have the following nucleicacid sequences: mGM-CSF polynucleotide cassette (SEQ ID NO: 21), UL49.5polynucleotide cassette (SEQ ID NO: 22), UL49.5-fs polynucleotidecassette (SEQ ID NO: 23), UL49.5/mGM-CSF polynucleotide cassette (SEQ IDNO: 24), and UL49.5-fs/mGM-CSF polynucleotide cassette (SEQ ID NO: 25)

As shown in FIG. 5, the plasmid pCMV-mGM-CSF encodes the mGM-CSF geneunder CMV promoter and BGH polyA control. The mGM-CSF ORF is flanked bya unique NheI restriction endonuclease site 5′ of the mGM-CSF startcodon and a unique XhoI site 3′ of the mGM-CSF stop codon and 5′ of theBGH polyA site. 3′ of the BGH polyA site are unique XbaI, BamHI and PacIrestriction endonuclease sites. 5′ of the CMV promoter is the upstreamportion of the α27 promoter region from the BlpI site to the upstreamα27 promoter terminus. Plasmids pNhe-UL49.5-Xho and pNhe-UL49.5-fs-Xhoencode the UL49.5 and UL49.5-fs ORFs flanked by NheI and XhoI sites inorder to facilitate replacement of the mGM-CSF ORF in pCMV-mGM-CSF withthe UL49.5 and UL49.5-fs ORFs. pEF1α-UL49.5 encodes the UL49.5 ORF undertranscriptional control of the EF1α promoter and SV40 late polyA signal.Upstream of the EF1α promoter are unique BamHI and PacI restrictionendonuclease sites. Downstream of the late SV40 polyA signal is a uniqueXbaI restriction endonuclease site. The plasmidpSPΔ34.5-flα27P-Us11-PacI is the targeting vector illustrated in FIGS.2, 3, and 4 and described in Example 2, above.

Using the above strategy, the following variant HSV were made andisolated, unless otherwise indicated:

-   -   1. OV-UL49.5: This variant HSV contains the same construct as        the OV-mGM-CSF construct, below, except the mGM-CSF open reading        frame is replaced with the BHV UL49.5 gene.    -   2. OV-UL49.5-fs: This variant HSV contains UL49.5 with a single        nucleotide addition between the second and third UL49.5 codons        to create a frameshift (fs) mutation.    -   3. OV-mGM-CSF: This variant HSV contains the mGM-CSF gene under        control of the CMV promoter. Transcription was terminated at the        BGHpA, as in OncoVEX^(GM-CSF).    -   4. OV-UL49.5/mGM-CSF: This variant HSV contains mGM-CSF under        CMV promoter and BGHpA control, as well as UL49.5 under control        of the EF1α promoter and SV40 late polyadenylation signal.    -   5. OV-UL49.5-fs/mGM-CSF: This variant HSV contains the same        construct as the OV-UL49.5/mGM-CSF construct except that the        UL49.5 fs mutation described above is incorporated. This variant        HSV is a good isogenic control for the contribution evasion of        CD8+ T-cells by UL49.5 makes in a viral background encoding        mGM-CSF. This variant HSV is expected to be isolated easily,        since OV-UL49.5/mGM-CSF was obtained.

Although the variant HSV prepared in this example were prepared from thewild-type HSV Strain Patton, which is known in the art [see, e.g.,International patent application publication no. WO 2002/06513; U.S.Pat. No. 4,818,694; and Mohr, I. et al. J Virol. 2001 June; 75(11):5189-5196, and U.S. Patent Application Publication No. 20060039894],those of ordinary skill in the art will appreciate that the materialsand techniques described herein may be used to prepare homologousvariants of other HSV strains, including, but not limited to, awild-type HSV Strain 17 having the nucleic acid sequence set forth inSEQ ID NO: 1. Predicted nucleic acid sequence of complete variants of anHSV Strain 17, that are homologous to the variant Patton HSV OV-UL49.5,OV-UL49.5-fs, OV-mGM-CSF, OV-UL49.5/mGM-CSF, and OV-UL49.5-fs/mGM-CSF,described above, are therefore set forth in SEQ ID NOS:26-30,respectively, as non-limiting, exemplary sequences of variant HSVaccording to this invention.

The targeting vectors were co-transfected into Vero cells with purifiedΔ34.5 viral DNA, and then recombinant viruses expressing IE-Us11 wereselected by growth on U251 cells, which are non-permissive for thegrowth of Δ34.5 viruses. Thus, IE-Us1 functioned as a dominantselectable marker in this system to select variant HSV successfullyencoding the polynucleotide cassettes.

To demonstrate the successful generation and isolation of variant HSVencoding the mGM-CSF, UL49.5, UL49.5-fs, or UL49.5/mGM-CSFpolynucleotide cassettes, viral DNA from high titer viral stocks derivedfrom isolated plaques was prepared and digested with two restrictionenzymes that cut outside the Δ34.5 loci and release fragments containingIEUs11 and the ectopic transgenic sequences for analysis by Southernblot. Digested DNA was separated on a 0.8% agarose gel, transferred to anylon membrane and probed with a labeled fragment that hybridizes to theΔ34.5 locus. As shown in FIG. 6, bands in each lane agree with thepredicted fragment sizes for each variant HSV generated, as well as thecontrol viruses Δ34.5 and OV-2711.

Example 4: Infectivity of Virus Encoding the UL49.5/GM-CSF Construct

This Example demonstrates that recombinant HSV-1 engineered to encodethe UL49.5/mGM-CSF construct described in Example 3, above, and as shownin FIG. 4, express the UL49.5 polypeptide, since the UL49.5 polypeptidewas detected in Vero cells infected with OV-UL49.5/mGM-CSF.

Vero cells were mock infected or infected with five separate plaquepurified isolates of OV-UL49.5/mGM-CSF at a multiplicity of infection(MOI) equal to 1. At 24 hours post-infection, the media was aspiratedand the cells were lysed in Laemmli's buffer. The lysate was then boiledto denature polypeptides, and the boiled samples were then separated bySDS-PAGE and Western blotted using an antibody raised against the UL49.5polypeptide (anti-H11 polyclonal rabbit antibody (described in LipinskaA D, et al. J Virol 2006; 80:5822-32). As shown in FIG. 7A, protein ofthe expected molecular weight, approximately 12 kDa, was observed invirally-infected cells but not in the mock-infected cells. Anon-specific ˜15 kDa background signal demonstrated that a similaramount of mock infected sample was present compared to the virallyinfected samples. This ruled out the possibility that the 12 kDa bandwas absent from the mock-infected lane because of lower-than-expectedmock sample concentration or gel loading.

Next, Vero cells were infected with either wild-type (WT) Patton strainHSV-1 or OV-UL49.5 at MOI=5, and, at 24 hours post-infection (PI),protein lysates were prepared and analyzed for UL49.5 polypeptideexpression by Western blot, as described above. UL49.5 polypeptideclearly accumulated to easily detectable levels in cells infected withOV-UL49.5, but was not detected in cells infected with WT HSV-1,indicating that recombinant HSV-1 that encode and express the UL49.5polypeptide (OV-UL49.5) was successfully generated (FIG. 7B).

Example 5: Detection of mGM-CSF mRNA in Mouse Balb/c Mammary 4T1 CancerCells Infected with BV-mM-CSF or BV-UL49.5/GM-CSF

This example demonstrates that recombinant HSV-1 engineered to encodethe mGM-CSF or UL49.5/GM-CSF construct described in Example 3, above,express mGM-CSF mRNA.

4T1 cells were either mock infected or infected with wild-type (WT), orthe OV-mGM-CSF or OV-UL49.5/mGM-CSF viruses at MOI=5. At 24 hourspost-infection, cells were lysed by addition of Trizol reagent (LifeTechnologies) and RNA was purified using RNeasy silica columns (Qiagen)according to the manufacturer's directions. Purified RNA was thentreated with DNase I (new England Biolabs) for 30 mins at 37° C., EDTAadded to a final concentration of 5 mM and heated for 15 mins at 75° C.to inactivate DNase I. qRT-PCR was then performed using SYBR Greendetection with oligonucleotide primers that detect mouse 18S rRNA,mGM-CSF mRNA or viral VP16 mRNA. 18s rRNA was detected using aproprietary pair of primers purchased from SA Biosciences (Valencia,Calif.). mGM-CSF mRNA was detected using primers mGM-CSF-FW(5′-CTGTCACCCGGCCTTGGAAGC-3′) (SEQ ID NO: 31) and mGM-CSF-RV(5′-ACAGGCATGTCATCCAGGAGGT-3′) (SEQ ID NO: 32). VP16 mRNA was detectedusing primers VP16-FW (5′-TCGGCGTGGAAGAAACGAGAGA-3′) (SEQ ID NO: 33) andVP16-RV (5′-CGAACGCACCCAAATCGACA-3′) (SEQ ID NO: 34).

mGM-CSF mRNA expression (normalized to 18S rRNA signal) was clearlydetected in cells infected with the recombinant OV-2711 HSV variantsencoding GM-CSF under CMV protocol control (OV-mGM-CSF andOV-UL49.5/mGM-CSF) (FIG. 8A). Furthermore, mGM-CSF mRNA expression wassimilar in cells infected with the single transgene insertion variant,OV-mGM-CSF compared to the double transgene insertion variant,OV-UL49.5/GM-CSF.

Next, the expression of VP16 mRNA (normalized to 18S rRNA signal) wasdetected. VP16 is an essential HSV-1 gene, so mRNA expression isdetected in all infected cells analyzed in this experiment. There was atmost a two-fold difference in VP16 mRNA expression among the virallyinfected cells (FIG. 8B). Absence of mGM-CSF mRNA detection compared todetection of VP6 message in WT-infected cells demonstrated that themGM-CSF signal detected (see FIG. 8A) was specific to cells infectedwith OV-2711 variants that encode an ectopic mGM-CSF expressioncassette.

Example 6: Evaluation of Variant HSV in an MBT-2 Bladder Cancer Model

This Example demonstrates that the OV-2711 virus described in Example 1can replicate in and spread through MBT-2 cell monolayers, which allowsfull evaluation of the contribution viral evasion of CD8+ T-cells makesto viral spread and anti-tumor efficacy. The experiments described inthis Example can also be used to evaluate the recombinant HSV virusesencoding the constructs described in Examples 2 and 3, above.

OV-2711, as well as a Δ34.5 and Δ34.5ΔICP47 viruses were evaluated in anin vitro model of bladder cancer using MBT-2 cells. Δ34.5ΔICP47 mimicsthe genetic arrangement of OncoVEX^(GM-CSF) that produces immediateearly expressed Us11 to overcome the protein synthesis block encounteredby Δ34.5 mutants.

Viruses were added to the media of replicate plates with adherent MBT-2cell monolayers at a multiplicity of infection equal to 0.01. Duplicatesets of plates to which the indicated viruses were added were frozen at2, 24 hours, 48, 72, 96 and 120 hours after viral addition. The plateswere then thawed, the media pipetted up and down on the plate surface todetach and homogenize all cells, transferred to a 15 ml conical tube,sonicated for 15 seconds in a sonicating water bath and frozen. Thetubes were then thawed and the level of infectious virus present in eachsample determined by plaque assay using Vero cell monolayers, which arepermissive for the replication and growth of Δ34.5 variants.

While Δ34.5 did not replicate and spread through MBT-2 monolayers,presumably due to the cellular block to protein synthesis observed inmany cancer cell lines infected with Δ34.5 variants, both OV-2711 andΔ34.5ΔICP47 grew nearly as well as wild-type (WT) (FIG. 8). Although itappeared that OV-2711 accumulated to lower titers than Δ34.5ΔICP47, theOV-2711 input dose was proportionally lower in this experiment, and isexpected to accumulate to similar titers as Δ34.5ΔICP47 when used at thesame input dose. It was clear from this experiment that OV-2711 canreplicate in and spread through MBT-2 cell monolayers. Therefore, thesyngeneic MBT-2 tumor model can accurately assess the role viral evasionof anti-HSV CD8+ T-cells plays in HSV1 oncolytic virus therapeuticefficacy, and can be used to characterize the properties of therecombinant viruses encoding the constructs described in Examples 2 and3, above.

The oncolytic viruses can also be tested as described in this Exampleusing a suitable melanoma, ovarian, glioma and/or other cancer celllines, in order to characterize the activity of the recombinantoncolytic viruses against cancer cells.

Example 7: Evaluation of Variant HSV in a Syngeneic, Immune-CompetentMurine Model of Bladder Cancer

The following experiments may be used to examine the performance ofoncolytic viruses described herein (e.g., in Examples 2 and 3, above),including variant HSV based on OV-2711 (e.g., those shown in FIGS. 3 and4), and to compare the performance of such viruses with other virusesknown in the art, such as OncoVEX^(GMCSF) and viruses similar thereto.The experiments demonstrate that functions known to preclude recognitionof infected cells by CD8+ T-cells will result in enhanced tumorreduction without compromising viral-mediated tumor antigen vaccination.

In addition to direct oncolysis, an immune-mediated componentcontributes to HSV-1 oncolytic virus efficacy in immune-competent mice.Using immune-competent mice with syngeneic, bilateral subcutaneous(s.c.) tumors, previous studies established that treatment of one tumorwith oncolytic virus induced regression of the treated and untreatedcontralateral tumor (see Toda M, et al. “Herpes simplex virus as an insitu cancer vaccine for the induction of specific anti-tumor immunity.”Hum Gene Ther 1999; 10:385-93). While treated and untreated tumors bothregressed, oncolytic virus was only detected in the treated tumor.Furthermore, regression of the uninjected, contralateral tumor resultedfrom an anti-tumor CD8+ T-cell response. A pre-existing host immuneresponse capable of neutralizing HSV-1, however, would likely limitoncolytic virus spread through the injected tumor and diminish theefficiency at which antitumor immunity develops.

These experiments compare the oncolytic and immune evasion properties ofthe variant HSV that are generated as described in Examples 2 and 3,above, in a syngeneic, immune-competent murine model of bladder cancer,in which the mice are seropositive for HSV-1. In particular, theexperiments demonstrate that HSV variants having intact endogenous Us11and Us12 genes, and lacking ICP34.5 encoding genes, wherein the ICP34.5encoding genes are replaced by IE-Us11 and UL49.5 (TAP inhibitor) or byIE-Us11, UL49.5 and GM-CSF (“OV-UL49.5” or “OV-UL49.5-GM-CSF”,respectively), are superior to HSV lacking immune evasion abilities(e.g., the ability to inhibit TAP or otherwise evade cytolytic CD8+ Tcell responses), such as OncoVEX^(GMCSF) or OV-2711 (although OV-2711encodes Us12 and is predicted to inhibit TAP function in human cells, itis defective in Us12 function during infection of rodent cells becausethe Us12 polypeptide exhibits significantly reduced affinity for andinhibitory activity against rodent TAP), because they can persist longerin the tumor and have greater capacity i) for direct oncolysis; and ii)to stimulate systemic anti-tumor immunity.

To compare the anti-tumor activity of variant HSV that evade CD8+T-cells (“OV-UL49.5” & “OV-UL49.5-GM-CSF”) in mice with those thatcannot (“OV-2711” & “OV-mGM-CSF”), immune-competent mice are firstimmunized with a non-neuorovirulent, replication competent HSV-1, or asub-lethal dose of WT virus, as described in Chahlavi A, et al. “Effectof prior exposure to herpes simplex virus 1 on viral vector-mediatedtumor therapy in immunocompetent mice.” Gene Ther (1999) 6:1751-8, tomimic HSV-1 seropositive humans or those that convert to sero-positivityafter oncolytic virus-treatment.

HSV-1⁺ immune response development is monitored by ELISA for theappearance of virus-neutralizing antibodies [see, Chahlavi A, et al.“Effect of prior exposure to herpes simplex virus 1 on viralvector-mediated tumor therapy in immunocompetent mice.” Gene Ther (1999)6:1751-8]. Three (3) months are allowed to pass in order to ensure thedevelopment of a memory immune response, at which time a set of mice arechallenged with virulent virus to prove that memory has been establishedphysiologically and functionally, by detecting presence of anti-HSV-1cytotoxic T lymphocytes (CTLs) [see, Kavanagh D G, Gold M C, Wagner M,Koszinowski U H, Hill A B. J Exp Med. 2001 Oct. 1; 194(7):967-78. Next,syngeneic s.c. MBT-2 mouse BC seed tumors are implanted bilaterally intoeach flank of HSV-1-vaccinated C3H/HeJ mice (Charles River BreedingLaboratories). The seed tumors are prepared by injecting 1×10⁸ MBT-2cells [see, Mickey D D, et al. (1982) J Urol. 127(6):1233-7] s.c. intothe flank of a BALB/c nu/nu outbred mouse (Charles River BreedingLaboratories). Tumors are measured every other day with Vernier calipersand their volume determined by using the formula0.52×width×height×depth. Once the tumor size reaches 150 mm³, the animalis euthanized, and the explanted tumor sectioned into 2×2×2 mmfragments. Individual tumor fragments are then surgically implanted s.c.into naïve mice. When tumors reach 50 mm³, a 50 μl solution of oncolyticvirus is injected directly into the tumor.

A s.c. tumor model has significant advantages for this experiment, as itallows precise, non-invasive tumor measurements over time; oncolyticvirus-treated and untreated tumor growth on different flanks can becompared directly; and untreated tumors on contralateral flanks can bedirectly monitored for viral antigens.

A range of HSV-variant doses can be tested in groups of 5HSV-1-vaccinated animals with 50 mm³ MBT-2 tumors implanted s.c. on bothflanks (Vero lysate, 10⁶, 10⁷, and 10⁸ pfu—20 mice). OV-2711 isadministered on days 1, 3 and 5, and tumor size measured as describedabove. Although this regimen with a 5×10⁶ pfu dose of 2711 eliminatesthe growth of tumors in nude mice, lower efficacy for OV-2711 isexpected in immune-competent HSV-1-vaccinated mice due to prematureclearance of 2711 by CD8+ T-cells and other components of the immunesystem. Despite the ability to inhibit TAP-mediated viral antigenpresentation, it is expected that inhibition is not absolute and a lowlevel of viral antigens are displayed on MHC-I. TAP inhibition simplyslows the rate at which the virus is cleared by CD8+ T-cells compared toa corresponding virus that is deficient in functions capable ofefficiently inhibiting TAP function. Therefore, higher doses are testedto determine the lowest viral dose that yields maximal tumor regression(lowest effective dose—LED). Tumor volume may be monitored as describedabove for up to 60 days.

Following establishment of the dynamic range of OV-2711 efficacy inimmune-competent HSV-1 vaccinated animals, the panel of variant HSVdescribed in Examples 2 and 3, and having the genetic modificationsshown in FIGS. 2, 3 and/or 4 are tested. The dose of infectious virus isexpected to be 10-fold below the OV-2711 LED. This should provide asuitable dynamic range to evaluate the contribution UL49.5 and mGM-CSFexpression make to increased oncolytic virus efficacy. Tumor volume maybe monitored over time as described above. Since oncolytic viruses donot spread to the uninjected contralateral tumor, the size of thecontralateral tumor is a good measure of the efficiency of oncolyticvirus-mediated tumor vaccination. Animals may be sacrificed when theyexhibit signs of excessive tumor burden or appear moribund, the wetweight of the tumors recorded, and the tumor tissue flash frozen andstored for future analysis to detect viral replication and CD8+ T-cellinfiltration by immunohistochemistry (IHC) with anti-HSV-1 and anti-CD8antibodies, respectively (as available, e.g., from Abcam® (Cambridge,Mass.) and Santa Cruz Biotechnology (Santa Cruz, Calif.)).

It is expected that: i) injected and uninjected tumors will regress, butvirus will only be detected in the injected tumor; and ii) regression ofthe uninjected, contralateral tumor results from an anti-tumor T-cellresponse. Results from these experiments will define how Us12-likeimmune-evasion functions contribute to oncolytic virus efficacy and thedevelopment of anti-tumor immunity. If CD8+ T-cell evasion contributesto oncolytic virus efficacy, a greater reduction in the size ofOV-UL49.5-injected and corresponding contralateral tumors compared totumors in mice treated with OV-2711 or OV-mGM-CSF is expected. It isalso expected that tumors injected with OV-UL49.5-GM-CSF, will haveenhanced APC recruitment by mGM-CSF coupled with improved viral spreadconferred by UL49.5, and that the OV-UL49.5-GM-CSF variant will prove tobe a superior oncolytic virus for the treatment of bladder cancer.

The oncolytic viruses describe above can also be tested as described inthis Example using suitable in vive animal models of melanoma, ovarian,glioma and/or other cancers, in order to characterize the activity ofthe recombinant oncolytic viruses against cancer cells. Such animalmodels are known in the art and described in detail herein, above.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A variant herpes simplex virus (HSV) having anintact endogenous Us12 encoding gene and an intact endogenous Us11encoding gene, lacking functional ICP34.5 encoding genes, wherein eachICP34.5 encoding gene is replaced by a polynucleotide cassettecomprising: (a) a Us11 encoding gene operably associated with animmediate early (TE) promoter; and (b) at least one heterologous geneencoding a polypeptide capable of enhancing an anti-tumor response. 2.The variant HSV according to claim 1, in which said at least oneheterologous gene encodes an immunomodulatory polypeptide.
 3. Thevariant HSV according to claim 2, in which the immunomodulatorypolypeptide is selected from the group consisting of a TAP 1/2 (“TAP”)inhibitor, granulocyte macrophage colony stimulating factor (GM-CSF),tumor necrosis factor (TNF)-alpha and CD40 ligand (CD40L).
 4. Thevariant HSV according to claim 3, in which said at least oneimmunomodulatory polypeptide is GM-CSF.
 5. The variant HSV according toclaim 1, in which said at least one heterologous gene encodes a prodrugconverting enzyme.
 6. The variant HSV according to claim 1, comprisingat least two (2) heterologous genes encoding a polypeptide capable ofenhancing an anti-tumor response.
 7. The variant HSV according to claim6, wherein said one of said two heterologous genes encodes a TAPinhibitor and the other encodes a mammalian GM-CSF.
 8. The variant HSVaccording to claim 7, in which the TAP inhibitor inhibits a non-humanTAP.
 9. The variant HSV according to claim 8, in which the TAP inhibitorinhibits a murine TAP.
 10. The variant HSV according to claim 9, inwhich the TAP inhibitor is the UL49.5 polypeptide from bovineherpesvirus.
 11. The variant HSV according to claim 6, wherein one ofsaid two heterologous genes encodes a TAP inhibitor and the otherencodes a prodrug converting enzyme.
 12. A variant herpes simplex virus(HSV) having an intact endogenous Us12 encoding gene and an intactendogenous Us11 encoding gene, lacking functional ICP34.5 encodinggenes, wherein each ICP34.5 encoding gene is replaced by apolynucleotide cassette comprising: (a) a Us11 encoding gene operablyassociated with an immediate early (IE) promoter; and (b) a geneencoding an inhibitor of antigen presentation on class I majorhistocompatibility complex (MHC) molecules, wherein said inhibitor iscapable of inhibiting antigen presentation on the surface of virallyinfected tumor cells.
 13. The variant HSV according to claim 12, whereinsaid inhibitor of antigen presentation is a TAP inhibitor.
 14. Thevariant HSV according to claim 12, in which the polynucleotide cassetteadditionally comprises a heterologous gene encoding a polypeptidecapable of enhancing an anti-tumor response.
 15. The variant HSVaccording to claim 14, in which the heterologous gene encodes GM-CSF.16. The variant HSV according to claim 1 or 12, in which the IE promoteris an α27 IE promoter.
 17. The variant HSV according to claim 1 or 12,in which the heterologous gene is operably associated with a promoterselected from the group consisting of a CMV promoter and an EF1αpromoter.
 18. The variant HSV according to claim 13, wherein said TAPinhibitor is a bovine herpesvirus (BHV) UL49.5 polypeptide.
 19. Avariant herpes simplex virus (HSV) having an intact endogenous Us12encoding gene and an intact endogenous Us11 encoding gene, lackingfunctional ICP34.5 encoding genes, wherein each ICP34.5 encoding gene isreplaced with a polynucleotide cassette comprising a nucleic acidsequence set forth in one of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24 and SEQ ID NO:
 25. 20. A variant herpes simplex virus(HSV) having a genome sequence set forth in SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO:
 30. 21. A pharmaceuticalformulation comprising: the variant HSV according to claim 1, 12, 19, or20, and a pharmaceutically acceptable carrier for administration totumor cells.
 22. The pharmaceutical formulation according to claim 21,wherein said administration to tumor cells is in situ.
 23. Thepharmaceutical formulation according to claim 21, wherein said virusselectively infects human bladder cancer cells, human melanoma cells,human ovarian cancer cells, or human glioblastoma cells.
 24. A methodfor killing tumor cells in a subject comprising: administering to asubject in need thereof the pharmaceutical formulation according toclaim 21 under conditions effective to kill tumor cells in the subject.25. The method according to claim 24, wherein the tumor cells areselected from the group consisting of astrocytoma, oligodendroglioma,meningioma, neurofibroma, glioblastoma, ependymoma, Schwannoma,neurofibrosarcoma, medulloblastoma, melanoma cells, pancreatic cancercells, prostate carcinoma cells, breast cancer cells, lung cancer cells,colon cancer cells, hepatoma cells, mesothelioma, bladder cancer cells,and epidermoid carcinoma cells.
 26. The method according to claim 24,wherein said virus selectively replicates in human bladder cancer cells,melanoma cells, ovarian cancer cells, or glioblastoma cells.
 27. Themethod according to claim 24, wherein the subject is a mammal.
 28. Themethod according to claim 24, wherein the mammal is a human.
 29. Themethod according to claim 24, wherein the administration is carried outby injection, infusion, instillation or inhalation.
 30. A method fortreating cancer, wherein said method comprises administering to anindividual in need of treatment, a therapeutically effective amount ofthe pharmaceutical formulation according to claim
 24. 31. The methodaccording to claim 30, wherein the individual is a mammal.
 32. Themethod according to claim 31, wherein the mammal is a human.
 33. Themethod according to claim 30, wherein the cancer is selected from thegroup consisting of bladder cancer, melanoma, ovarian cancer andglioblastoma.